Anti-IgE antibodies

ABSTRACT

The present invention relates to a method for adjusting the affinity of a polypeptide to a target molecule by a combination of steps, including: (1) the identification of aspartyl residues which are prone to isomerization; (2) the substitution of alternative residues and screening the resulting mutants for affinity against the target molecule In a preferred embodiment, the method of subtituting residues is affinity maturation with phage display (AMPD). In a further preferred embodiment the polypeptide is an antibody and the target molecule is an antigen. In a further preferred embodiment, the antibody is anti-IgE and the target molecule is IgE. In another embodiment, the invention relates to an anti-IgE antibody having improved affinity to IgE.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of U.S. Ser. No. 09/920,171, filed Aug. 1,2001, which is a continuation of U.S. Ser. No. 09/296,005, filed Apr.21, 1999, now U.S. Pat. No. 6,290,957, which is a continuation of U.S.Ser. No. 08/887,352, filed Jul. 2, 1997, now U.S. Pat. No. 5,994,511,all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to immunoglobulin E (IgE), IgEantagonists, anti-IgE antibodies capable of binding to human IgE, and toa method of improving polypeptides, including anti-IgE antibodies.

[0003] IgE is a member of the immunoglobulin family that mediatesallergic responses such as asthma, food allergies, type 1hypersensitivity and the familiar sinus inflammation suffered on awidespread basis. IgE is secreted by, and expressed on the surface ofB-cells or B-lymphocytes. IgE binds to B-cells (as well as to monocytes,eosinophils and platelets) through its Fc region to a low affinity IgEreceptor, known as FcεRII. Upon exposure of a mammal to an allergen,B-cells bearing a surface-bound IgE antibody specific for the antigenare “activated” and developed into IgE-secreting plasma cells. Theresulting allergen-specific IgE then circulates through the bloodstreamand becomes bound to the surface of mast cells in tissues and basophilsin the blood, through the high affinity receptor also known as FcεRI.The mast cells and basophils thereby become sensitized for the allergen.Subsequent exposure to the allergen causes a cross linking of thebasophilic and mast cellular FcεRI which results in a release ofhistamine, leukotrienes and platelet activating factors, eosinophil andneutrophil chemotactic factors and the cytokines IL-3, IL-4, IL-5 andGM-CSF which are responsible for clinical hypersensitivity andanaphylaxis.

[0004] The pathological condition hypersensitivity is characterized byan excessive immune response to (an) allergen(s) resulting in grosstissue changes if the allergen is present in relatively large amounts orif the humoral and cellular immune state is at a heightened level.

[0005] Physiological changes in anaphylactic hypersensitivity caninclude intense constriction of the bronchioles and bronchi of thelungs, contraction of smooth muscle and dilation of capillaries.Predisposition to this condition, however, appears to result from aninteraction between genetic and environmental factors. Commonenvironmental allergens which induce anaphylactic hypersensitivity arefound in pollen, foods, house dust mites, animal danders, fungal sporesand insect venoms. Atopic allergy is associated with anaphylactichypersensitivity and includes the disorders, e.g., asthma, allergicrhinitis and conjunctivitis (hay fever), eczema, urticaria and foodallergies. However, anaphylactic shock, a dangerous life-threateningcondition anaphylaxis is usually provoked by insect stings or parentalmedication.

[0006] Recently, a treatment strategy has been pursued for Type 1hypersensitivity or anaphylactic hypersensitivity which attempts toblock IgE from binding to the high-affinity receptor (FcεRI) found onbasophils and mast cells, and thereby prevent the release of histamineand other anaphylactic factors resulting in the pathological condition.

[0007] WO 93/04173, published Mar. 4, 1993 describes human IgE/IgG1chimeras wherein IgG1 residues are substituted for the analogous IgEresidues. Applicants' copending application U.S. Ser. No. 08/405,617describes humanized anti-IgE antibodies wherein a murine antibodydirected against human IgE (MaE11) was used to provide the CDR regionswhich were substituted into an IgG1 immunoglobulin framework (rhuMaE25).A technique of humanization is described in Reichman, L. et al., (1988)Nature 332: 323 and in Jones, P. T. et al. (1986), Nature 321: 522.

[0008] While humanization of murine antibodies has been established toprovide anti-IgE molecules which provide similar affinity to IgE asmurine MaE11 without the immunogenic response elicited by the latter(Shields et al., (1995) Int. Arch. Allergy Immunol. 107: 308-312), ithas still not resulted in the construction of an anti-IgE with affinityfor IgE which is decidedly better than MaE11 or a murine anti-IgE.

[0009] Recombinant monoclonal antibodies are subject to degradationreactions that affect all polypeptides or proteins, such asisomerization of aspartic acid and asparagine residues. As shown in Fig.A, below, aspartate residues (I) in -Asp-Gly- sequences can isomerize toisoaspartate (III) through a cyclic imide intermediate (II). (Geiger &Clarke, J. Biol. Chem. 262: 785-794 (1987)). The carboxylic acid sidechain of the aspartic acid (I) reacts with the amide nitrogen of theadjacent glycine to form a cyclic aspartic acid intermediate (II) whichthen forms into an -isoaspartic acid-glycine- residue (III). Theequilibrium, rate, and pH dependence of this reaction have been studiedin model peptides separated by reversed phase high performance liquidchromatography. (Oliyai & Borchardt, Pharm Res. 10, 95-102 (1993)). Thetendency to undergo isomerization is believed to also depend upon thelocal flexibility of the portion of the molecule containing the-Asp-Gly- sequence (Geiger & Clarke, supra).

[0010] An example of a known antibody which undergoes aspartic acidisomerization is the potent anti-IgE antibody known as rhuMabE-25 (E25).This event may occur spontaneously, but can be induced to occur when E25is incubated at 37° C. for 21 days. The end result is the insertion ofan additional methyl group into the polypeptide backbone of theantibody, which can result in conformational changes and reduction inbinding affinity. A study of E25 with -c-Asp-Gly- and -iso-Asp-Gly-variants at position VL 32-33 indicated that while the isomerizationevent can be minimized by substitution of alanine or glutamic acid forresidue VL32, the substitution itself results in a three-fold reductionin binding. Cacia et al., supra.

[0011] Thus, there exists a great need for the creation of improvedpolypeptides, including antibodies, which not only don't exhibit the“deactivating” event of aspartyl isomerization, but also displayaffinity to the target molecule (e.g., antigen) equal to or greater thanthe unimproved polypeptide's affinity.

SUMMARY

[0012] The present invention relates to a method for improving apolypeptide having affinity to a target molecule by a combination ofsteps, including: (1) the identification of aspartyl residues which areprone to isomerization; (2) the substitution of alternative residues andscreening the resulting mutants for affinity against the targetmolecule. In a preferred embodiment, the method of substituting residuesis affinity maturation with phage display (AMPD). In a further preferredembodiment the polypeptide is an antibody and the target molecule is anantigen. In a further preferred embodiment, the antibody is anti-IgE andthe target molecule is IgE.

[0013] In an even more preferred embodiment, the invention relates to amethod for improving the affinity of the anti-IgE antibody E25 byreplacement of VL CDR-L1 residue 32Asp with Glu, along with themodification of VL CDR-L1 residues 27Gln, 28 Ser and 31Tyr to Lys, Proand Gly, respectively. In an even more preferred embodiment, the E25anti-IgE antibody has additional modifications at residues VH CDR2:53Thr to Lys, 55Asp to Ser, 57Ser to Glu and 59Asn to Lys.

[0014] In another embodiment, the invention relates to an anti-IgEantibody having improved affinity to IgE.

[0015] In a preferred embodiment, the anti-IgE antibody comprises heavyand light chain residues comprising the sequence fragments labeled “E27”and “E26” in FIG. 2. Alternatively, the anti-IgE antibody comprises thefull length heavy and light chain sequences labeled “E27” and “E26” inFIG. 12.

[0016] The present invention also relates to a composition of improvedaffinity anti-IgE or functional fragments thereof having pharmaceuticalutility. The present invention also relates to an article of manufacturecomprising an improved affinity anti-IgE antibody.

[0017] In yet another embodiment, the present invention relates to amethod of reducing or inhibiting the IgE-mediated production ofhistamine.

[0018] In yet another embodiment, the present invention also relates toa method of treating an IgE-mediated disorder by the administration ofthe antibodies of the invention or functional fragments thereof.

[0019] Other aspects of the invention will become apparent from thefollowing detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a comparison of the VH and VL domains between the murineantibody MAE11, human consensus sequences of heavy chain subgroup III(humIII) and light chain κ subgroup I (humκI) and fragment F(ab)-2, amodified human antibody fragment with CDR residues and certain frameworkresidues modified to murine.

[0021]FIG. 2 is a sequence comparison of the differences between thelight chain and heavy chain CDR domains between rhuMabE25, E426, andsequences E26 and E27. The residue numbering here is consecutive, asopposed to that of Kabat et al. Also note that these sequences are onlyfragments and not the actual full-length heavy and light chain residues.

[0022]FIG. 3 is a graph of an FACS-based assay indicating the ability ofthe tested antibody to inhibit FITC-conjugated IgE binding to theα-chain of the high-affinity FcεRI receptor expressed on CHO 3D10 cells.The percentage of inhibition by murine mAb MaE11 (□), the negativecontrol humanized mAb4D5 (▪), F(ab)-2 (∘), F(ab)-9 (), F(ab)-11 (Δ) andF(ab)-12 (▴) are represented. The data points are the average of threeexperiments, except for mAb 4D5, which is a single experimental value.The results indicate that MaE11 and the tested F(ab)s block FITC-IgEbinding to CHO 3D10 cells expressing FcεRI α-chain.

[0023]FIG. 4 is a graph of an FACS-based assay measuring the binding ofthe tested antibody to IgE-loaded with the α-subunit of thehigh-affinity receptor FcεRI expressed on CHO 3D10 cells. The percentagebinding by murine mAb MaE11 (∘), humanized variant 12 (▴), positivecontrol murine mAb MaE1 (), negative control antibody murine MOPC21(Δ), and negative control humanized mAb4D5 (□). On an arithmetic/linearscale, mean channel fluorescence values at 0.1 μg/ml were MPOC21 7.3,MaE1 32.1, MaE11 6.4, hu4D5 4.7 and huMaE11 4.6. All three murine mAbswere murine isotype IgG1, and both humanized mAbs were human isotypeIgG1. Data points are the average of three experiments. The resultsindicate that MaE11 and F(ab)-12 do not bind to IgE-loaded CHO 3D10cells expressing FcεRI U-chain.

[0024]FIG. 5 is a graph of the molar ratio of anti-IgE v. percentinhibition of ragweed-induced histamine release. E25 () and E26 (∘) areshown. The results indicate that the F(ab) form of E26 has superiorinhibition of ragweed-induced histamine release in a dose dependentmanner with a half-maximal inhibition molar ratio of 44:1(anti-IgE:RSIgE).

[0025]FIG. 6 is a graphical representation of the affinity enrichmentafter various rounds of affinity selections described in part II ofExample 4. The ratio of binding enrichment for each pool to that of thewild-type (Emut/Ewt) is displayed. The results indicate that the VLlibraries (represented by “a” and “b”) displayed successively improvedrelative enrichments, up to about 10-fold greater than wild-type after5-6 rounds of enrichment. Moreover, the VH libraries “c” and “d”)exhibited about a 3-fold improvement after around 3 rounds. Note that“a” corresponds to the Fab-phage library mutated at VL CDR-1 residues27, 28, 30 and 31, while “b” corresponds to mutations at 30, 31, 32 and34, while “c” and “d” are independent F(ab) libraries with mutations atresidues 101, 102, 103, 105 and 107.

[0026]FIG. 7 is a graph of the observed optical density vs.concentration of IgE competitor antibody in a phage ELISA competitionstudy of the final variants from combinations of the VL CDR1 mutationsin E26 with the VH CDR2 mutations in clones 235-5.1, 235-5.2, 235-5.3and 235-5.4, renamed E27, E695, E696 and E697, respectively, describedin part V of Example 4.

[0027]FIG. 8 is a graph of the absorbance at 490 nm of variousconcentration levels of E25, E26 and E27 anti-IgE antibody in the biotinplate assay described in part VI of Example 4.

[0028]FIG. 9 indicates the F(ab) apparent binding affinity of E25, E26and E27, as measured by BIAcore TM-2000 surface plasmon resonancesystem. 1.5 serial dilutions of F(ab) antibody fragments were injectedover the IgE chip in PBS/Tween buffer (0.05% Tween-20 in phosphatebuffered saline) at 25° C. using a flow rate of 20 μl/min. Theequilibrium dissociation constants (Kd) shown were calculated from theratio of observed kon/koff for each Fab variant.

[0029] FIGS. 10A-F is a sequence listing of the plasmid p426 which wasused as the template for the construction of library-specific stoptemplates in Example 4.

[0030]FIG. 11A is a diagram of plasmid pDH188 insert containing the DNAencoding the light chain and heavy chain (variable and constantdomain 1) of the Fab humanized antibody directed to the HER-2 receptor.VL and VH are the variable regions for the light and heavy chains,respectively. C_(k) is the constant region of the human kappa lightchain. CH1_(G1) is the first constant region of the human gamma 1 chain.Both coding regions start with the bacterial stII signal sequence.

[0031]FIG. 11B is a schematic diagram of the entire plasmid pDH188containing the insert described in 11A. After transformation of theplasmid into E. coli SR101 cells and the addition of helper phage, theplasmid is packaged into phage particles. Some of these particlesdisplay the Fab-p III fusion (where p III is the protein encoded by theM13 gene III DNA).

[0032]FIG. 12 represents the full-length heavy and light chain residuesof anti-IgE antibodies E25, E26 and E27.

[0033]FIG. 13 represents F(ab) fragments of anti-IgE antibodies E26 andE27.

[0034]FIG. 14 represents sFv fragments of anti-IgE antibodies E26 andE27.

[0035]FIG. 15 represents F(ab′)₂ fragments of anti-IgE antibodies E26and E27.

[0036] SEQ ID NO: 1 represents the sequence of the expression plasmidE426 used in the invention, also indicated in FIG. 10.

[0037] SEQ ID NO: 2 represents the variable heavy chain sequence ofMaE11 indicated in FIG. 1

[0038] SEQ ID NO: 3 represents the variable heavy chain sequence ofF(ab)-2 indicated in FIG. 1.

[0039] SEQ ID NO: 4 represents the variable heavy chain sequence ofhumIII indicated in FIG. 1.

[0040] SEQ ID NO: 5 represents the variable light chain sequence ofMaE11 indicated in FIG. 1.

[0041] SEQ ID NO: 6 represents the variable light chain sequence ofF(ab)-2 indicated in FIG. 1.

[0042] SEQ ID NO: 7 represents the variable light chain sequence ofhumIII indicated in FIG. 1.

[0043] SEQ ID NO: 8 represents the variable light chain sequence of E26and E27 indicated in FIG. 2.

[0044] SEQ ID NO: 9 represents the variable light chain sequence of E426indicated in FIG. 2.

[0045] SEQ ID NO: 10 represents the variable light chain sequence of E25indicated in FIG. 2.

[0046] SEQ ID NO: 11 represents the variable heavy chain sequence of E27indicated in FIG. 2.

[0047] SEQ ID NO: 12 represents the variable heavy chain sequence ofE25, E26 and E426 indicated in FIG. 2.

[0048] SEQ ID NO: 13 represents the full length variable light chainsequence of E25 as indicated in FIG. 12.

[0049] SEQ ID NO: 14 represents the full length heavy chain sequence ofE25 as indicated in FIG. 12

[0050] SEQ ID NO: 15 represents the full length light chain sequence ofE26 as indicated in FIG. 12.

[0051] SEQ ID NO: 16 represents the full length heavy chain sequence ofE26 as indicated in FIG. 12.

[0052] SEQ ID NO: 17 represents the full length light chain sequence ofE27 as indicated in FIG. 12.

[0053] SEQ ID NO: 18 represents the full length heavy chain sequence ofE27 as indicated in FIG. 12.

[0054] SEQ ID NO: 19 represents the variable light chain Fab fragment ofE26 and E27 as indicated in FIG. 12.

[0055] SEQ ID NO: 20 represents the variable heavy chain Fab fragment ofE26 as indicated in FIG. 13.

[0056] SEQ ID NO: 21 represents the variable heavy chain Fab fragment ofE27 as indicated in FIG. 13.

[0057] SEQ ID NO: 22 represents the sFv fragment of E26 as indicated inFIG. 14.

[0058] SEQ ID NO: 23 represents the sFv fragment of E27 as indicated inFIG. 14.

[0059] SEQ ID NO: 24 represents the variable light chain F(ab′)₂fragment for E26 and E27 as indicated in FIG. 15.

[0060] SEQ ID NO: 25 represents the variable heavy chain F(ab′)₂fragment for E26 as indicated in FIG. 15.

[0061] SEQ ID NO: 26 represents the variable heavy chain F(ab′)₂fragment for E27 as indicated in FIG. 15.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The mention of particular references, patent application andpatents throughout this application should be read as being incorporatedby reference into the text of the specification.

[0063] Definitions:

[0064] Terms used throughout this application are to be construed withordinary and typical meaning to those of ordinary skill in the art.However, Applicants desire that the following terms be given theparticular definition as defined below:

[0065] The terms “protein” or “polypeptide” are intended to be usedinterchangeably. They refer to a chain of two (2) or more amino acidswhich are linked together with peptide or amide bonds, regardless ofpost-translational modification (e.g., glycosylation orphosphorylation). Antibodies are specifically intended to be within thescope of this definition.

[0066] The polypeptides of this invention may comprise more than onesubunit, where each subunit is encoded by a separate DNA sequence.

[0067] The phrase “substantially identical” with respect to an antibodypolypeptide sequence shall be construed as an antibody exhibiting atleast 70%, preferably 80%, more preferably 90% and most preferably 95%sequence identity to the reference polypeptide sequence. The term withrespect to a nucleic acid sequence shall be construed as a sequence ofnucleotides exhibiting at least about 85%, preferably 90%, morepreferably 95% and most preferably 97% sequence identity to thereference nucleic acid sequence. For polypeptides, the length of thecomparison sequences will generally be at least 25 amino acids. Fornucleic acids, the length will generally be at least 75 nucleotides.

[0068] The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software(e.g., Sequence Analysis Software Package, Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Ave.,Madison, Wis. 53705). This software matches similar sequences byassigning degrees of homology to various substitutions, deletions, andother modifications.

[0069] The term “antibody” is used in the broadest sense, andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments (e.g., Fab,F(ab′)₂ and Fv) so long as they exhibit the desired biological activity.Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

[0070] Native antibodies and immunoglobulins are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges. Each heavy chain hasat one end a variable domain (VH) followed by a number of constantdomains. Each light chain has a variable domain at one end (VL) and aconstant domain at its other end. The constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains (Clothia etal., J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl.Acad. Sci. U.S. Pat. No. 82,4592-4596 (1985).

[0071] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of the environment in whichit was produced. Contaminant components of its production environmentare materials which would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, theantibody will be purified as measurable by at least three differentmethods: 1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight; 2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequentator; or 3)to homogeneity by SDS-PAGE under reducing or non-reducing conditionsusing Coomasie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0072] A “species-dependent antibody” e.g., a mammalian anti-human IgEantibody, is an antibody which has a stronger binding affinity for anantigen from a first mammalian species than it has for a homologue ofthat antigen from a second mammalian species. Normally, thespecies-dependent antibody “bind specifically” to a human antigen (i.e.,has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second non-human mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody.

[0073] The term “antibody mutant” refers to an amino acid sequencevariant of an antibody wherein one or more of the amino acid residueshave been modified. Such mutant necessarily have less than 100% sequenceidentity or similarity with the amino acid sequence having at least 75%amino acid sequence identity or similarity with the amino acid sequenceof either the heavy or light chain variable domain of the antibody, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, and most preferably at least 95%. Since the method of theinvention applies equally to both polypeptides, antibodies and fragmentsthereof, these terms are sometimes employed interchangeably.

[0074] The term “variable” in the context of variable domain ofantibodies, refers to the fact that certain portions of the variabledomains differ extensively in sequence among antibodies and are used inthe binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) also known ashypervariable regions both in the light chain and the heavy chainvariable domains. There are at least two techniques for determiningCDRs: (1) an approach based on cross-species sequence variability (i.e.,Kabat et al., Sequences of Proteins of Immunological Interest (NationalInstitute of Health, Bethesda, Md. 1987); and (2) an approach based oncrystallographic studies of antigen-antibody complexes (Chothia, C. etal. (1989), Nature 342: 877). With respect to Applicants' anti-IgEantibody, certain CDRs were defined by combining the Kabat et al. andChothia et al. approaches. The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat etal.) The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector function, such asparticipation of the antibody in antibody-dependent cellular toxicity.

[0075] The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments.

[0076] Papain digestion of antibodies produces two identical antigenbinding fragments, called the Fab fragment, each with a single antigenbinding site, and a residual “Fc” fragment, so-called for its ability tocrystallize readily.

[0077] Pepsin treatment yields an F(ab′)₂ fragment that has two antigenbinding fragments which are capable of cross-linking antigen, and aresidual other fragment (which is termed pFc′). Additional fragments caninclude diabodies, linear antibodies, single-chain antibody molecules,and multispecific antibodies formed from antibody fragments. As usedherein, “functional fragment” with respect to antibodies, refers to Fv,F(ab) and F(ab′)₂ fragments.

[0078] An “Fv” fragment is the minimum antibody fragment which containsa complete antigen recognition and binding site. This region consists ofa dimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

[0079] The Fab fragment [also designated as F(ab)] also contains theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxyl terminus of the heavy chainCH1 domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains have a free thiol group. F(ab′)fragments are produced by cleavage of the disulfide bond at the hingecysteines of the F(ab′)₂ pepsin digestion product. Additional chemicalcouplings of antibody fragments are known to those of ordinary skill inthe art.

[0080] The light chains of antibodies (immunoglobulin) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino sequences of theirconstant domain.

[0081] Depending on the amino acid sequences of the constant domain oftheir heavy chains, “immunoglobulins” can be assigned to differentclasses. There are at least five (5) major classes of immunoglobulins:IgA, IgD, IgE, IgG and IgM, and several of these may be further dividedinto subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1and IgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called α, δ, ε, γ and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.The preferred immunoglobulin for use with the present invention isimmunoglobulin E.

[0082] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In additional to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein, Nature 256, 495 (1975), or may be made byrecombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. Themonoclonal antibodies for use with the present invention may also beisolated from phage antibody libraries using the techniques described inClackson et al. Nature 352: 624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222: 581-597 (1991).

[0083] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567);Morrison et al. Proc. Natl. Acad. Sci. 81, 6851-6855 (1984).

[0084] “Humanized” forms of non-human (e.g. murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321,522-525 (1986); Reichmann et al., Nature 332, 323-329 (1988) and Presta,Curr. Op. Struct. Biol. 2, 593-596 (1992).

[0085] “Single-chain Fv” or “sFv” antibody fragments comprise the VH andVL domains of an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the sFvto form the desired structure for antigen binding. For a review of sFv,see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0086] The term “diabodies” refers to a small antibody fragments withtwo antigen-binding sites, which fragments comprise a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

[0087] The phrase “functional fragment or analog” of an antibody is acompound having qualitative biological activity in common with afull-length antibody. For example, a functional fragment or analog of ananti-IgE antibody is one which can bind to an IgE immunoglobulin in sucha manner so as to prevent or substantially reduce the ability of suchmolecule from having the ability to bind to the high affinity receptor,FcεRI.

[0088] The term “amino acid” and “amino acids” refer to all naturallyoccuring L-α-amino acids. The amino acids are identified as hereinafterdescribed under section A. Antibody Preparation: (iv) Generation ofmutant antibodies. The term “amino acid variant” refers to moleculeswith some differences in their amino acid sequences as compared to anative amino acid sequence.

[0089] “Substitutional” variants are those that have at least one aminoacid residue in a native sequence removed and a different amino acidinserted in its place at the same position. The substitutions may besingle, where only one amino acid in the molecule as been substituted,or they may be multiple, where two or more amino acids have beensubstituted in the same molecule. “Insertional” variants are those withone or more amino acids inserted immediately adjacent to an amino acidat a particular position in a native sequence. Immediately adjacent toan amino acid means connected to either the α-carboxyl or α-aminofunctional group of the amino acid. “Deletional” variants are those withone or more amino acids in the native amino acid sequence removed.Ordinarily, deletional variants will have one or two amino acids deletedin a particular region of the molecule.

[0090] The term “cell”, “cell line” and “cell culture” are usedinterchangeably, and all such designations include progeny. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological property, as screened for in theoriginally transformed cell, are included.

[0091] The “host cells” used in the present invention generally areprokaryotic or eukaryotic hosts. Examples of suitable host cells aredescribed in Section B. Vectors, Host Cells and Recombinant Methods:(vii) Selection and transformation of host cells.

[0092] “Transformation” means introducing DNA into an organism so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integration.

[0093] “Transfection” refers to the taking up of an expression vector bya host cell whether or not any coding sequences are in fact expressed.

[0094] The terms “transfected host cell” and “transformed” refer to theintroduction of DNA into a cell. The cell is termed “host cell” and itmay be either prokaryotic or eukaryotic. Typical prokaryotic host cellsinclude various strains of E. coli. Typical eukaryotic host cells aremammalian, such as Chinese hamster ovary or cells of human origin. Theintroduced DNA sequence may be from the same species as the host cell ora different species from the host cell, or it may be a hybrid DNAsequence, containing some foreign and some homologous DNA.

[0095] The terms “replicable expression vector” and “expression vector”refer to a piece of DNA, usually double-stranded, which may haveinserted into it a piece of foreign DNA. Foreign DNA is defined asheterologous DNA, which is DNA not naturally found in the host cell. Thevector is used to transport the foreign or heterologous DNA into asuitable host cell. Once in the host cell, the vector can replicateindependently of the host chromosomal DNA and several copies of thevector and its inserted (foreign) DNA may be generated.

[0096] The term “vector” means a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of the DNA in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites, and sequences which control thetermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may in some instances, integrateinto the genome itself. In the present specification, “plasmid” and“vector” are sometimes used interchangeably, as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other form of vector which serve equivalentfunction as and which are, or become, known in the art. Typicalexpression vectors for mammalian cell culture expression, for example,are based on pRK5 (EP 307,247), pSV16B (WO 91/08291), and pVL1392(Pharmingen).

[0097] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as the antibody mutants disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

[0098] The expression “control sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers.

[0099] An “isolated” nucleic acid molecule is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the antibody nucleic acid. An isolated nucleic acid moleculeis other than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguishable from thenucleic acid molecule as it exists in natural cells. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

[0100] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. This can bea gene and a regulatory sequence(s) which are connected in such a way asto permit gene expression when the appropriate molecules (e.g.,transcriptional activator proteins) are bound to the regulatorysequences(s). For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it affects the transcription ofthe sequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0101] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented.

[0102] A “disorder” is any condition that would benefit from treatmentwith the polypeptide. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question.

[0103] The term “immunosuppressive agent” as used herein for adjuncttherapy refers to substances that act to suppress or mask the immunesystem of the host into which a graft is being transplanted. This wouldinclude substances that suppress cytokine production, downregulate orsuppress self-antigen expression, or mask the MHC antigens. Examples ofsuch agents include 2-amino-5-aryl-5-substituted pyrimidines (See U.S.Pat. No. 4,665,077), azathioprine (or cyclophosphamide, in case ofadverse reaction to azathioprine); bromocryptine; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and NHC fragments;cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone,methylprednisone, and dexamethasone; cytokine or cytokine receptorantagonists including anti-interferon-γ, -β, or α-antibodies; anti-tumornecrosis factor-α antibodies; anti-tumor necrosis factor-β antibodies;anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; solublepeptide containing a LFA-3 binding domain (WO 90/08187 published Jul.26, 1990); streptokinase; TGF-β; streptodomase; RNA or DNA from thehost; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S.Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science251: 430-432 (1991); WO 90/11294; and WO 91/01133); and T cell receptorantibodies (EP 340,109) such as T10B9. These agents are administered atthe same time or at separate times from CD11a antibody, and are used atthe same or lesser dosages than as set forth in the art. The preferredadjunct immunosuppressive agent will depend on many factors, includingthe type of disorder being treated including the type of transplantationbeing performed, as well as the patient's history, but a general overallpreference is that the agent be selected from cyclosporin A, aglucocorticosteroid (most preferably prednisone or methylprednisolone),OKT-3 monoclonal antibody, azathioprine, bromocryptine, heterologousanti-lymphocyte globulin, or a mixture thereof.

[0104] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

[0105] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including human, domestic and farm animals,nonhuman primates, and zoo, sports, or pet animals, such as dogs,horses, cats, cows, etc.

[0106] The term “epitope tagged” when used herein refers to polypeptidefused to an “epitope tag.” The epitope tag polypeptide has enoughresidues to provide an epitope against which an antibody thereagainstcan be made, yet is short enough such that it does not interfere withactivity of the polypeptide. The epitope tag preferably also is fairlyunique so that the antibody thereagainst does not substantiallycross-react with other epitopes. Suitable tag polypeptide generally haveat least 6 amino acid residues and usually between about 8-50 amino acidresidues (preferably between about 9-30 residues). Examples include theflu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell.Biol. 8: 2159-2165 (1988))); the c-myc tag and the 8F9, 3C7, 6E10, G4,B7 and 9E10 antibodies thereagainst (Evan et al, Mol Cell. Biol. 5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering 3(6): 547-553(1990)). In certain embodiments, the epitope tag is a “salvage receptorbinding epitope.”

[0107] As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁,IgG₂, IgG₃ or IgG₄) that is responsible for increasing the in vivo serumhalf-life of the IgG molecule.

[0108] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fungal, plantor animal origin, or fragments thereof.

[0109] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdrimycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside{“Ara-C”),Cyclophosphamide, thiotepa, Taxotere (docetaxel), Bulsulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin,Aminopterin, Dactinomycin, Mitomycine, Esperamicins (see U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards.

[0110] The term “prodrug” as used in this application refers to aprecursor or derivative from of a pharmaceutically active substance thatis less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in CancerChemotherapy,” Biochemical Society Transactions, 14, pp. 375-382, 615Meeting, Belfast (1986) and Stella et al., (ed.), “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardtet al, (ed.), pp. 247-267, Human Press (1985). The prodrugs of thisinvention include, but are not limited to phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, α-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

[0111] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody. The label may itself be detectable (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

[0112] As used herein, “solid phase” means a non-aqueous matrix to whichthe antibody of the present invention can adhere. Examples of solidphases encompassed herein include those formed partially or entirely ofglass (e.g. controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

[0113] As used herein, anti-human IgE antibody means an antibody whichbinds to human IgE in such a manner so as to inhibit or substantiallyreduce the binding of such IgE to the high affinity receptor, FcεRI.Preferably this anti-IgE antibody is E25.

[0114] As used herein, the term “IgE-mediated disorder” means acondition or disease which is characterized by the overproduction and/orhypersensitivity to the immunoglobulin IgE. Specifically it should beconstrued to include conditions associated with anaphylactichypersensitivity and atopic allergies, including for example: asthma,allergic rhinitis and conjunctivitis (hay fever), eczema, urticaria andfood allergies. However, the serious physiological condition ofanaphylactic shock, usually caused by bee or snake stings or parentalmedication is also encompassed under the scope of this term.

[0115] As used herein, “affinity maturation using phage display” (AMPD)refers to a process described in Lowman et al., Biochemistry 30(45):10832-10838 (1991), see also Hawkins et al., J. Mol. Biol.254: 889-896(1992). While not strictly limited to the following description, thisprocess can be described briefly as: several hypervariable region sites(e.g. 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibody mutants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage expressing the various mutants can be cycled through rounds ofbinding selection, followed by isolation and sequencing of those mutantswhich display high affinity. The method is also described in copendingapplication U.S. Ser. No. 08/050,058, filed Apr. 30, 1993, now U.S. Pat.No. 5,750,373, issued May 12, 1998. A modified procedure involvingpooled affinity display is described in Cunningham, B. C. et al., EMBOJ. 13(11), 2508-2515 (1994).

[0116] The method provides a method for selecting novel bindingpolypeptides comprising: a) constructing a replicable expression vectorcomprising a first gene encoding a polypeptide, a second gene encodingat least a portion of a natural or wild-type phage coat protein whereinthe first and second genes are heterologous, and a transcriptionregulatory element operably linked to the first and second genes,thereby forming a gene fusion encoding a fusion protein; b) mutating thevector at one or more selected positions within the first gene therebyforming a family of related plasmids; c) tranforming suitable host cellswith the plasmids; d) infecting the transformed host cells with a helperphage having a gene encoding the phage coat protein; e) culturing thetransformed infected host cells under conditions suitable for formingrecombinant phagemid particles containing at least a portion of theplasmid and capable of transforming the host, the conditions adjusted sothat no more than a minor amount of phagemid particles display more thanone copy of the fusion protein on the surface of the particle; f)contacting the phagemid particles with a target molecule so that atleast a portion of the phagemid particles bind to the target molecule;and g) separating the phagemid particles that bind from those that donot. Preferably, the method further comprises transforming suitable hostcells with recombinant phagemid particles that bind to the targetmolecule and repeating steps d) through g) one or more times.

[0117] Alternatively, the method includes polypeptides which arecomposed of more than one subunit, wherein the the replicable expressionvector comprising a transcription regulatory element operably linked toDNA encoding the subunit of interest is fused to the phage coat protein.

[0118] As used herein, the term “antibody phage library” refers to thephage library used in the affinity maturation process described aboveand in Hawkins et al., J. Mol. Biol.254: 889-896 (1992), and in Lowmanet al., Biochemistry 30(45): 10832-10838 (1991). Each library comprisesa hypervariable region (e.g. 6-7 sites) for which all possible aminoacid substitutions are generated. The antibody mutants thus generatedare displayed in a monovalent fashion from filamentous phage particlesas fusions to the gene III product of M13 packaged within each particleand expressed on the exterior of the phage.

[0119] As used herein, “room” or “ambient temperature” shall be 23°C.-25° C.

[0120] As used herein “binding polypeptide” means any polypeptide thatbinds with a selectable affinity to a target molecule. Preferably, thepolypeptide will be a protein that most preferably contains more thanabout 100 amino acid residues. Typically, the polypeptide will be ahormone or an antibody or a fragment thereof.

[0121] As used herein, “high affinity” means an affinity constant (Kd)of <10⁻⁵ M and preferably <10⁻⁷ M under physiological conditions.

[0122] As used herein, “target molecule” means any molecule, notnecessarily a protein, for which it is desirable to produce an antibodyor ligand. Preferably, however, the target will be a protein and mostpreferably the target will be an antigen. However, receptors, such as ahormone receptors should particularly be included within the scope ofthis term.

[0123] As used herein, all numbering of immunoglobulin amino acidresidues, including the amino acid numbering of peptides correspondingto specific portions of IgE, mutant IgE molecules and chimeric IgEmolecules that appears herein is done according to the immunoglobulinamino acid residue numbering system of Kabat et al., Sequences ofProteins of Immunological Interest (National Institute of Health,Bethesda, Md. 1987).

[0124] Modes for Carrying out the Invention

[0125] I. Method of Improving Target Molecule Affinity.

[0126]

[0127] A. Identification of Isomerizable Aspartyl Residues.

[0128] In practicing the present invention, the identification ofisomerizable aspartyl residues prone to isomerization can be effected byany technique known to those of ordinary skill in the art. For example,Cacia et al., Biochemistry 35, 1897-1903 (1996), describe a processwherein the anti-IgE antibody E25 (which contains -Asp-Gly- residues) isincubated at 37° C. for 21 days. The identification of isomerized-Asp-Gly- were effected by chromatographic and mass spectrometricanalysis of untreated and protease treated fragments. Sinceisomerization has also been reported to occur with asparaginyl residues(T. Geiger and S. Clarke, J. Biol. Chem. 262(2), 785-794 (1987), thepresent invention may also be preferably practiced to the systematicevaluation and improvement of polypeptides containing asparaginylresidues.

[0129] B. Selection of Alternate Residues Which Improve Target MoleculeAffinity.

[0130] Many techniques are available to one of ordinary skill in the artwhich permit the optimization of receptor affinity. Typically, thesetechniques all involve substitution of various amino acid residues atthe site of interest, followed by a screening analysis of receptoraffinity of the mutant polypeptide. A technique preferred for use withthe present invention is affinity maturation using phage display(Hawkins et al. J. Mol. Biol.254: 889-896 (1992); Lowman et al.,Biochemistry 30(45): 10832-10838 (1991)). Briefly, several hypervariableregion sites (e.g., 6-7 sites) are mutated to generate all possibleamino acid substitutions at each site. The antibody mutants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage expressing the various mutants can be cycled throughrounds of binding selection, followed by isolation and sequencing ofthose mutants which display high affinity.

[0131] The method of selecting novel binding polypeptides preferablyutilizes a library of structurally related polypeptides. The library ofstructurally related polypeptides, fused to a phage coat protein, isproduced by mutagenesis, and preferably, a single copy of each relatedpolypeptide is displayed on the surface of the phagemid particlecontaining DNA encoding that polypeptide. These phagemid particles arethen contacted with a target molecule and those particles having thehighest affinity for the target are separated from those of loweraffinity. The high affinity binders are then amplified by infection of abacterial host and the competitive binding step is repeated. The processis repeated until polypeptides of the desired affinity are obtained.

[0132] Alternatively, multivalent phage (McCafferty et al. (1990),Nature 348, 552-554; Clackson et al. (1991), Nature 352, 624-628) canalso be used to express random point mutations (generated by use of anerror-prone DNA polymerase) to generate a library of phage antibodyfragments which could then be screened by affinity to antigen. Hawkinset al, (1992) J. Mol. Biol. 254: 889-896.

[0133] Preferably during the affinity maturation process, the replicableexpression vector is under tight control of the transcription regulatoryelement, and the culturing conditions are adjusted so that the amount ornumber of phagemid particles displaying more than one copy of the fusionprotein on the surface of the particle is less than about 1%. Alsopreferably, the amount of phagemid particles displaying more than onecopy of the fusion protein is less than 10% the amount of phagemidparticles displaying a single copy of the fusion protein. Mostpreferably the amount is less than 20%.

[0134] Typically, in the method of this invention, the expression vectorwill further contain secretory signal sequence(s) fused to the DNAencoding each subunit of the polypeptide, and the transcriptionregulatory element will be a promoter system. Preferred promoter systemsare selected from: LacZ, λ_(PL), TC, T7 polymerase, tryptophan, andalkaline phosphatase promoters and combinations thereof.

[0135] Also typically, the first gene will encode a mammalian protein,preferably, the protein will be an anti-IgE antibody. Additionalantibodies are exemplified in section II.A. Antibody preparation, (vi)multispecific antibodies (note however, that antibodies need not bemultispecific). Additional polypeptides include human growth hormone(hGH), N-methionyl human growth hormone, bovine growth hormone,parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain,proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (THS), and leutinizing hormone (LH), glycoprotein hormonereceptors, calcitonin, glucagon, factor VIII, lung surfactant,urokinase, streptokinase, human tissue-type plasminogen activator(t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumornecrosis factor-alpha and -beta, enkephalinase, human serum albumin,mullerian-inhibiting substance, mouse gonadotropin-associated peptide, amicrobial protein, such as betalactamase, tissue factor protein,inhibin, activin, vascular endothelial growth factor, receptors forhormones or growth factors, integrin, thrombopoietin, protein A or D,rheumatoid factors, nerve growth factors such as NGF-β, platelet-growthfactor, transforming growth factors (TGF) such as TGF-alpha andTGF-beta, insulin-like growth factor-I and —II, insulin-like growthfactor binding proteins, CD-4, DNase, latency associated peptide,erythropoietin, osteoinductive factors, interferons such asinterferon-alpha, -beta and -gamma, colony stimulating factors (CSFs)such as M-CSF, GM-CSF and G-CSF, interleukins (Ils) such as IL-1, IL-2,IL-3, IL-4, superoxide dismutase, decay accelerating factor, viralantigen, HIV envelope proteins such as GP120, GP140, atrial natriureticpeptides A, B or C, immunoglobulins, and fragments of any of theabove-listed proteins.

[0136] Preferably, the first gene will encode a polypeptide of one ormore subunits containing more than about 100 amino acid residues andwill be folded to form a plurality of rigid secondary structuresdisplaying a plurality of amino acids capable of interacting with thetarget. Preferably the first gene will be mutated at codonscorresponding to only the amino acids capable of interacting with thetarget so that the integrity of the rigid secondary structures will bepreserved.

[0137] Normally, the method of this invention will employ a helper phageselected from: M13KO7, M13R408, M13-VCS, and Phi X 174. The preferredhelper phage is M13KO7, and the preferred coat protein is the M13 Phagegene II coat protein. The preferred host is E. coli, and proteasedeficient strains of E. coli. Novel hGH variants selected by the methodof the present invention have been detected. Phagemid expression vectorswere constructed that contain a suppressible termination codonfunctionally located between the nucleic acids encoding the polypeptideand the phage coat protein.

[0138] 1. Choice of Polypeptides for Display on the Surface of a Phage.

[0139] Repeated cycles of “polypeptide” selection are used to select forhigher and higher affinity binding by the phagemid selection of multipleamino acid changes which are selected by multiple selection of cycles.Following a first round of phagemid selection, involving a first regionof selection of amino acids in the ligand or antibody polypeptide,additional rounds of phagemid selection in other regions or amino acidsof the ligand are conducted. The cycles of phagemid selection arerepeated until the desired affinity properties are achieved. Toillustrate this process, Example 4 phage display was conducted incycles. Pooled affinity, combination of mutations from different CDRs,etc.

[0140] From the foregoing, it will be appreciated that the amino acidresidues that form the binding domain of the polypeptide will not besequentially linked and may reside on different subunits of thepolypeptide. That is, the binding domain tracks with particularsecondary structure at the binding site and not the primary structure.Thus, generally, mutations will be introduced into codons encoding aminoacids within a particular secondary structure at sites directed awayfrom the interior of the polypeptide so that they will have thepotential to interact with the target.

[0141] However, there is no requirement that the polypeptide chosen as aligand or antibody to a target molecule normally bind to that target.Thus, for example, a glycoprotein hormone such as TSH can be chosen as aligand for the FSH receptor and a library of mutant TSH molecules areemployed in the method of this invention to produce novel drugcandidates.

[0142] This invention thus contemplates any polypeptide that binds to atarget molecule, particularly antibodies. Preferred polypeptides arethose that have pharmaceutical utility. Example antibodies are recitedin section II. A. Antibody preparation (iv) multispecific antibodies(Note that antibodies need not be multispecific). More preferredpolypeptides include: growth hormone, including human growth hormone,des-N-methionyl human growth hormone, and bovine growth hormone;parathyroid hormone; thyroid stimulating hormone; thyroxine; insulinA-chain; insulin B-chain; prorelaxin; mouse gonadotropin-associatedpeptide; a microbial protein, such as betalactamase; tissue factorprotein; inhibin; activin; vascular endothelial growth factor; receptorsfor hormones or growth factors; integrin; thrombopoietin; protein A orD; rheumatoid factors; nerve growth factor such NGF-β; platelet-derivedgrowth factor; fibroblast growth factor such as aFGF and bFGF, epidermalgrowth factor; transforming growth factor (TGF) such as TGF-alpha andTGF-beta; insulin-like growth factor-I and —II; insulin-like growthfactor binding proteins; CD-4; DNase; latency associated peptide;erythropoietin; osteoinductive factors; such as, for example, a portionof the HIV envelope; immunoglobulins; and fragments of any of theabove-listed polypeptides. In addition, one or more predetermined aminoacid residues on the polypeptide may be substituted, inserted, ordeleted, for example, to produce products with improved biologicalproperties. Further, fragments of these polypeptides, especiallybiologically active fragments, are included. Yet more preferredpolypeptides of this invention are human growth hormone, and atrialnatriuretic peptides A, B and C, endotoxin, subtilisin, trypsin andother serine proteases Also preferred as polypeptide hormones that canbe defined as any amino acid sequence produced in a first cell thatbinds specifically to a receptor on the same cell type (autocrinehormones) or a second cell type (non-autocrine) and caused aphysiological response characteristic of the receptor-bearing cell.Among such polypeptide hormones are cytokines, lymphokines, neurotrophichormones and adenohypophyseal polypeptide hormones such as growthhormone, prolactin, placental lactogen, luteinizing hormone,follicle-stimulating hormone, β-lipotropin, γ-lipotropin and theendorphins; hypothalamic release-inhibiting hormone such ascorticotropin-release factors, growth hormone release-inhibitinghormone, growth hormone-release factor; and other polypeptide hormonessuch as atrial natriuretic peptides A, B or C.

[0143] 2. Obtaining a First Gene (Gene 1) Encoding the DesiredPolypeptide.

[0144] The gene encoding the desired polypeptide (e.g., antibody) can beobtained my methods known in the art (see generally, Sambrook et al.,Molecular Biology: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., (1989)). If the sequence of the gene is known, theDNA encoding the gene may be chemically synthesized (Merrifield, J. Am.Chem. Soc. 85: 2149 (1963)). If the sequence of the gene is not known,or if the gene has not previously been isolated, it may be cloned from acDNA library (made from RNA obtained from a suitable tissue in which thedesired gene is expressed) or from a suitable genomic DNA library. Thegene is then isolated using an appropriate probe. For cDNA libraries,suitable probes include monoclonal or polyclonal antibodies (providedthat the cDNA library is an expression library), oligonucleotides, andcomplementary or homologous cDNAs or fragments thereof. The probes thatmay be used to isolate the gene of interest from genomic DNA librariesinclude cDNAs or fragments thereof that encode the same or a similargene, homologous genomic DNAs or DNA fragments, and oligonucleotides.Screening the cDNA or genomic library with the selected probe isconducted using standard procedures as described in chapters 10-12 ofSambrook et al., supra.

[0145] An alternative means to isolating the gene encoding thepolypeptide (e.g. antibody) of interest is to use polymerase chainreaction methodology (PCR) as described in section 14 of Sambrook etal., supra. This method requires the use of oligonucleotides that willhybridize to the gene of interest, thus, at least some of the DNAsequence for this gene must be known in order to generate theoligonucleotides.

[0146] After the gene has been isolated, it may be inserted into asuitable vector (preferably a plasmid) for amplification, as describedgenerally in Sambrook et al., supra.

[0147] 3. Constructing Replicable Expression Vectors.

[0148] While several types of vectors are available and may be used topractice this invention, plasmid vectors are the preferred vectors foruse herein, as they may be constructed with relative ease, and can bereadily amplified. Plasmid vectors generally contain a variety ofcomponents including promoter, signal sequences, phenotypic selectiongenes, origin of replication sites, and other necessary components asare known to those of ordinary skill in the art.

[0149] Promoters most commonly used in prokaryotic vectors include thelac Z promoter system, the alkaline phosphatase pho A promoter, thebacteriophage λPL promoter (a temperature sensitive promoter), the tacpromoter (a hybrid trp-lac promoter that is regulated by the lacrepressor), the tryptophan promoter, and the bacteriophage T7 promoter.For general descriptions of promoters, see section 17 of Sambrook et al,supra. While these are the most commonly used promoters, other suitablemicrobial promoters may be used as well.

[0150] Preferred promoters for practicing this invention are those thatcan be tightly regulated such that expression of the fusion gene can becontrolled. If expression is uncontrolled, leading to multiple copies ofthe fusion protein on the surface of the phagemid, there could bemultipoint attachment of the phagemid with the target. This multipointattachment, also called “avidity” or “chelate effect” is believed toresult in the selection of false “high affinity” polypeptides caused bymultiple copies of the fusion protein being displayed on the phagemidparticle in close proximity to one another in a manner as to “chelate”the target. When multipoint attachment occurs, the effective or apparentKd may be as high as the product of the individual Kds for each copy ofthe displayed fusion protein.

[0151] Through tight regulation of the expression of the fusion proteinsuch that no more than aminor amount, i.e., fewer than about 1%, of thephagemid particles contain multiple copies of the fusion protein, the“chelate effect” is overcome allowing proper selection of high affinitypolypeptides. Thus, depending on the promoter, culturing conditions ofthe host are adjusted to maximize the number of phagemid particlescontaining a single copy of the fusion protein and minimize the numberof phagemid particles containing multiple copies of the fusion protein.

[0152] Preferred promoters used to practice this invention are the lac Zpromoter and the pho A promoter. The lac Z promoter is regulated by thelac repressor protein lac i, and thus transcription of the fusion genecan be controlled by manipulation of the level of the lac repressorprotein. By way of illustration, the phagemid containing the lac Zpromoter is grown in a cell strain that contains a copy of the lac irepressor gene, a repressor for the lac Z promoter. Exemplary cellstrains containing the lac i gene include JM 101 and XL-1 blue. In thealternative, the host cell can be cotransfected with a plasmidcontaining both the repressor lac i and lac Z promoter. Occasionallyboth of the above techniques are used simultaneously, that is, phagemidparticles containing the lac Z promoter are grown in cell strainscontaining the lac i gene and the cell strains are cotransfected with aplasmid containing both the lac Z and lac i genes. Normally when onewishes to express a gene, to the transfected host above one would add aninducer such as isopropylthiogalactoside (IPTG). In the presentinvention however, this step is omitted to (a) minimize the expressionof the gene III fusions per phagemid number) and to (b) prevent poor orimproper packaging of the phagemid caused by inducers such as IPTG evenat low concentrations. Typically, when no inducer is added, the numberof fusion proteins per phagemid particle is above 0.1 (number of bulkfusion proteins number of phagemid particles). The most preferredpromoter used to practice this invention is pho A. This promoter isbelieved to be regulated by the level of inorganic phosphate in the cellwhere the phosphate acts to down-regulate the activity of the promoter.Thus, by depleting cells of phosphate, the activity of the promoter canbe increased. The desired result is achieved by growing cells in aphosphate enriched medium such as 2YT or LB thereby controlling theexpression of the gene III fusion.

[0153] One other useful component of vectors used to practice thisinvention is a signal sequence. This sequence is typically locatedimmediately 5′ to the gene encoding the fusion protein, and will thus betranscribed at the amino terminus of the fusion protein. However, incertain cases, the signal sequence has been demonstrated to be locatedat positions other than 5′ to the gene encoding the protein to besecreted. This sequence targets the protein to which it is attachedacross the inner membrane of the bacterial cell. The DNA encoding thesignal sequence may be obtained as a restriction endonuclease fragmentfrom any gene encoding a protein that has a signal sequence. Suitableprokaryotic signal sequences may be obtained from genes encoding, forexample, LamB or OmpF (Wong et al, Gene 68; 193 (1983)), MalE, PhoA andother genes. A preferred prokaryotic signal sequences for practicingthis invention is the E. coli heat-stable enterotoxin II(STII) signalsequence as described by Chang et al., Gene 55: 189 (1987)).

[0154] Another useful component of the vectors used to practice thisinvention is phenotypic selection genes. Typical phenotypic selectiongenes are those encoding proteins that confer antibiotic resistance uponthe host cell. By way of illustration, the ampicillin resistance gene(amp), and the tetracycline resistance (tet) are readily employed forthis purpose.

[0155] Construction of suitable vectors comprising the aforementionedcomponents as well as the gene encoding the described polypeptide(gene 1) are prepared using standard recombinant DNA procedures asdescribed in Sambrook et al., supra. Isolated DNA fragments to becombined to form the vector are cleaved, tailored, and ligated togetherin a specific order and orientation to generate the desired vector.

[0156] The DNA is cleaved using the appropriate restriction enzyme orenzymes in a suitable buffer. In general, about 0.2-1 μg of plasmid orDNA fragments is used with about 1-2 units of the appropriaterestriction enzyme in about 20 μl of buffer solution. Appropriatebuffers, DNA concentrations, and incubation times and temperatures arespecified by the manufacturers of the restriction enzymes. Generally,incubation times of about one or two hours at 37° C. are adequate,although several enzymes require higher temperatures. After incubation,the enzymes and other contaminants are removed by extraction of thedigestion solution with a mixture of phenol and chloroform, and the DNAis recovered from the aqueous fraction by precipitation with ethanol.

[0157] To ligate the DNA fragments together to form a functional vector,the ends of the DNA fragments must be compatible with each other. Insome cases, the ends will be directly compatible after endonucleasedigestion. However, it may be necessary to first convert the sticky endscommonly produced by endonuclease digestion to blunt ends to make themcompatible for ligation. To blunt the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with 10 units of theKlenow fragment of DNA polymerase I (Klenow) in the presence of the fourdeoxynucleotide triphosphates. The DNA is then purified byphenol-chloroform extraction and ethanol precipitation.

[0158] The cleaved DNA fragments may be size-separated and selectedusing DNA gel electrophoresis. The DNA may be electrophoresed througheither an agarose or a polyacrylamide matrix. The selection of thematrix will depend on the size of the DNA fragments to be separated.After electrophoresis, the DNA is extracted from the matrix byelectroelution, or, if low-melting agarose has been used as the matrix,by melting the agarose and extracting the DNA from it, as described insections 6.30-6.33 of Sambrook et al., supra.

[0159] The DNA fragments that are to be ligated together (previouslydigested with the appropriate restriction enzymes such that the ends ofeach fragment to be ligated are compatible) are put in solution in aboutequimolar amounts. The solution will also contain ATP, ligase buffer anda ligase such as T4 DNA ligase at about 10 units per 0.5 μg of DNA. Ifthe DNA fragment is to be ligated into a vector, the vector is at firstlinearized by cutting with the appropriate restriction endonuclease(s).The linearized vector is then treated with alkaline phosphatase or calfintestinal phosphatase. The phosphatasing prevents self-ligation of thevector during the ligation step.

[0160] After ligation, the vector with the foreign gene now inserted istransformed into a suitable host cell. Prokaryotes are the preferredhost cells for this invention. Suitable prokaryotic host cells includeE. coli strain M101, E. coli K12 strain 294 (ATCC number 31,446), E.coli strain W31 10 (ATCC number 27,325), E. coli X1776 (ATCC number31,537), E. coli XL-1 Blue (stratagene), and E. coli B; however, manyother strains of E. coli, such as HB11, NM522, NM538, NM539, and manyother species and genera of prokaryotes may be used as well. In additionto the E. coli strains listed above, bacilli such as Bacillus subtilisother enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species may all be used as hosts.

[0161] Transformation of prokaryotic cells is readily accomplished usingthe calcium chloride method as described in section 1.82 of Sambrook etal, supra. Alternatively, electroporation (Neumann et ai., EMBO J. 1:841 (1982)) may be used to transform these cells. The transformed cellsare selected by growth on an antibiotic, commonly tetracycline (tet) orampicillin (amp), to which they are rendered resistant due to thepresence of tet and/or amp resistance genes on the vector.

[0162] After selection of the transformed cells, these cells are grownin culture and the plasmid DNA (or other vector with the foreign geneinserted) is then isolated. Plasmid DNA can be isolated using methodsknown in the art. Two suitable methods are the small scale preparationDNA and the large-scale preparation of DNA as described in sections1.25-1.33 of Sambrook et al., supra. The isolated DNA can be purified bymethods known in the art such as that described in section 1.40 ofSambrook et al., supra. This purified plasmid DNA is then analyzed byrestriction mapping and/or DNA sequencing. DNA sequencing is generallyperformed by either the method of Messing et al., Nucleic Acids Res. 9:309 (1981) or by the method of Maxam et al., Meth. Enzymol. 65: 499(1980).

[0163] 4. Gene fusion.

[0164] The phage affinity step of the present invention contemplatesfusing the gene enclosing the desired polypeptide (gene 1) to a secondgene (gene 2) such that a fusion gene is generated during transcription.Gene 2 is typically a coat protein gene of a phage, and preferably it isthe phage M13 gene III coat protein, or a fragment thereof. Fusion ofgenes 1 and 2 may be accomplished by inserting gene 2 into a particularsite on a plasmid that contains gene 1, or by inserting gene 1 into aparticular site on a plasmid that contains gene 2.

[0165] Insertion of a gene into a plasmid requires that the plasmid becut at the precise location that the gene is to be inserted. Thus, theremust be a restriction endonuclease site at this location (preferably aunique site such that the plasmid will only be cut at a single locationduring restriction endonuclease digestion). The plasmid is digested,phosphatased, and purified as described above. The gene is then insertedinto this linearized plasmid by ligating the two DNAs together. Ligationcan be accomplished if the ends of the plasmid are compatible with theends of the gene to be inserted. If the same restriction enzymes is usedto cut both the plasmid and isolate the gene to be inserted, the DNAscan be ligated together directly using a ligase such as bacteriophage T4DNA ligase and incubating the mixture at 16° C. for 1-4 hours in thepresence of ATP and ligase buffer as described in section 1.68 ofSambrook et al., supra. If the ends are not compatible, they must firstbe made blunt by using the Klenow fragment of DNA polymerase I orbacteriophage T4 DNA polymerase, both of which require the fourdeoxyribonucleotide triphosphates to fill-in overhanging single-strandedends of the digested DNA. Alternatively, the ends may be blunted using anuclease such as nuclease S1 or mung-bean nuclease, both of whichfunction by cutting back the overhanging single strands of DNA. The DNAis then relegated using a ligase as described above. In some cases, itmay not be possible to blunt the ends of the gene to be inserted, as thereading frame of the coding region will be altered. To overcome thisproblem, oligonucleotide linkers may be used. The linkers serve as abridge to connect the plasmid to the gene to be inserted. These linkerscan be made synthetically as double stranded or single-stranded DNAusing standard methods. The linkers have one end that is compatible withthe ends of the gene to be inserted; the linkers are first ligated tothis gene using ligation methods described above. The other end of thelinkers is designed to be compatible with the plasmid for ligation. Indesigning the linkers, care must be taken to not destroy the readingframe of the gene to be inserted or the reading frame of the genecontained on the plasmid. In some cases, it may be necessary to designthe linkers such that they code for part of an amino acid, or such thatthey encode for one or more amino acids.

[0166] Between gene 1 and gene 2, DNA encoding a termination codon maybe inserted, such termination codons are UAG (amber), UAA (ocher) andUGA (opel), Microbiology, Davis et al., Harper & Row, New York, 1980, pp237, 245-47 and 274). The termination codon expressed in a wild typehost cell results in the synthesis of the gene 1 protein product withoutthe gene 2 protein attached. However, growth in a suppressor host cellresults in the synthesis of detectable quantities of fused protein. Suchsuppressor host cells contain a tRNA modified to insert an amino acid inthe termination codon position of the mRNA thereby resulting inproduction of detectable amounts of the fusion protein. Such suppressorhost cells are well known and described, such as E. coli suppressorstrain (Bullock et al., BioTechnologies 5, 376-379 (1987)). Anyacceptable method may be used to place such a termination codon into themRNA encoding the fusion polypeptide.

[0167] The suppressible codon may be inserted between the first geneencoding a polypeptide, and a second gene encoding at least a portion ofa phage coat protein. Alternatively, the suppressible termination codonmay be inserted adjacent to the fusion site by replacing the last aminoacid triplet in the polypeptide or the first amino acid in the phagecoat protein. When the phagemid containing the suppressible codon isgrown in a suppressor host cell, it results in the detectable productionof a fusion polypeptide containing the polypeptide and the coat protein.When the phagemid is grown in a non-suppressor host cell, thepolypeptide is synthesized substantially without fusion to the phagecoat protein due to termination at the inserted suppressible tripletencoding UAG, UAA or UGA. In the non-suppressor cell the polypeptide issynthesized and secreted from the host cell due to the absence of thefused phage coat protein which otherwise anchored it to the host cell.

[0168] 5. Alteration (mutation) of Gene 1 at Selected Positions.

[0169] Gene 1, encoding the desired polypeptide, may be altered at oneor more selected codons. However, the codon corresponding to theisomerizable aspartyl residue must be changed. An alteration is definedas a substitution, deletion, or insertion of one or more codons in thegene encoding the polypeptide that results in a change in the amino acidsequence of the polypeptide as compared with the unaltered or nativesequence of the same polypeptide. Preferably, the alterations will be bysubstitution of at least one amino acid with any other amino acid in oneor more regions of the molecule. The alterations may be produced by avariety of methods known in the art. These methods include but are notlimited to oligonucleotide-mediated mutagenesis and cassettemutagenesis.

[0170] a. Oligonucleotide-Mediated Mutagenesis

[0171] Oligonucleotide-mediated mutagenesis is the preferred method forpreparing substitution, deletion, and insertion variants of gene 1. Thistechnique is well known in the art as described by Zoller et al.,Nucleic Acids Res. 10: 6487-6504 (1987). Briefly, gene 1 is altered byhybridizing an oligonucleotide encoding the desired mutation to a DNAtemplate, where the template is the single-stranded form of the plasmidcontaining the unaltered or native DNA sequence of gene 1. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template which will thus incorporate theoligonucleotide primer, and will code for the selected alteration ofgene 1.

[0172] Generally, oligonucleotides of at least 25 nucleotides in lengthare used. An optimal oligonucleotide will have 12 to 15 nucleotides thatare completely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. USA 75: 5765 (1978).

[0173] The DNA template can only be generated by those vectors that areeither derived from bacteriophage M13 vectors (the commonly availableM13 mp18 and M13 mp19 vectors are suitable), or those vectors thatcontain a single-stranded phage origin or replication as described byViera et al., Meth. Enzymol. 153: 3 (1987). Thus, the DNA that is to bemutated must be inserted into one of these vectors in order to generatea single-stranded template. Production of the single-stranded templateis described in sections 4.21-4.41 of Sambrook et al., supra.

[0174] To alter the native DNA sequence, the oligonucleotide ishybridized to the single stranded template under suitable hybridizationconditions. A DNA polymerizing enzyme, usually the Klenow fragment ofDNA polymerase I, is then added to synthesize the complementary strandof the template using the oligonucleotide as a primer for synthesis. Aheteroduplex molecule is thus formed such that one strand of DNA encodesthe mutated form of gene 1, and the other strand (the original template)encodes the native, unaltered sequence of gene 1. This heteroduplexmolecule is then transformed into a suitable host cell, usually aprokaryote such as E. coli JM-101. After growing the cells, they areplated onto agarose plates and screened using the oligonucleotide primerradiolabelled with ³²-Phosphate to identify the bacterial colonies thatcontain the mutated DNA.

[0175] The method described immediately above may be modified such thata homoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (Amersham). This mixture isadded to the template-oligonucleotide complex. Upon addition of DNApolymerase to this mixture, a strand of DNA identical to the templateexcept for the mutated bases is generated. In addition, this new strandof DNA will contain dCTP-(aS) instead of dCTP, which serves to protectit from restriction endonuclease digestion. After the template strand ofthe double-stranded heteroduplex is nicked with an appropriaterestriction enzyme, the template strand can be digested with ExoIIInuclease or another appropriate nuclease past the region that containsthe site(s) to be mutagenized. The reaction is then stopped to leave amolecule that is only partially single-stranded. A completedouble-stranded DNA homoduplex is then formed using DNA polymerase inthe presence of all four deoxyribonucleotide triphosphates, ATP, and DNAligase. This homoduplex molecule can then be transformed into a suitablehost cell such as E. coli JM101, as described above.

[0176] Mutants with more than one amino acid to be substituted may begenerated in one of several ways. If the amino acids are located closetogether in the polypeptide chain, they may be mutated simultaneouslyusing one oligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom each other (separated by more than about ten amino acids), it ismore difficult to generate a single oligonucleotide that encodes all ofthe desired changes. Instead, one or two alternative methods may beemployed.

[0177] In the first method, a separate oligonucleotide is generated foreach amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. The alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants:wild-type DNA is used for the template, and oligonucleotide encoding thefirst desired amino acid substitution(s) is annealed to this template,and the heteroduplex DNA molecule is then generated. The second round ofmutagenesis utilizes the mutated DNA produced in the first round ofmutagenesis as the template. Thus, this template already contains one ormore mutations. The oligonucleotide encoding the additional desiredamino acid substitution(s) is then annealed to this template, and theresulting strand of DNA now encodes mutations from both the first andsecond rounds of mutagenesis. This resultant DNA can be used as atemplate in a third round of mutagenesis, and so on.

[0178] b. Cassette Mutagenesis

[0179] This method is also a preferred method for preparingsubstitution, deletion, and insertion variants of gene 1. The method isbased on that described by Wells et al. Gene 34: 315 (1985). Thestarting material is the plasmid (or other vector) comprising gene 1,the gene to be mutated. The codon(s) in gene 1 to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in gene 1. After the restriction sites have beenintroduced into the plasmid, the plasmid is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3′ and 5′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence of gene 1.

[0180] 6. Obtaining DNA Encoding the Desired Protein.

[0181] In an alternative embodiment, this invention contemplatesproduction of variants of a desired protein containing one or moresubunits. Each subunit is typically encoded by separate genes. Each geneencoding each subunit can be obtained by methods known in the art (see,for example, Section II). In some instances, it may be necessary toobtain the gene encoding the various subunits using separate techniquesselected from any of the methods described in Section II.

[0182] When constructing a replicable expression vector where theprotein of interest contains more than one subunit, all subunits can beregulated by the same promoter, typically located 5′ to the DNA encodingthe subunit, or each may be regulated by the same promoter, typicallylocated 5′ to the DNA encoding the subunits, or each may be regulated bya separate promoter suitably oriented in the vector so that eachpromoter is operably linked to the DNA it is intended to regulate.Selection of promoters is carried out as described in Section III above.

[0183] In constructing a replicable expression vector containing DNAencoding the protein of interest having multiple subunits, the reader isreferred to FIG. 11, where, by way of illustration, a vector isdiagrammed showing DNA encoding each subunit of an antibody fragment.This figure shows that, generally, one of the subunits of the protein ofinterest will be fused to a phage coat protein such as M13 gene III.This gene fusion generally will contain its own signal sequence. Aseparate gene encodes the other subunit or subunits, and it is apparentthat each subunit generally has its own signal sequence. FIG. 11 alsoshows that a single promoter can regulate the expression of bothsubunits. Alternatively, each subunit may be independently regulated bya different promoter. The protein of interest subunit-phage coat proteinfusion construct can be made as described in Section IV above.

[0184] When constructing a family of variants of the desiredmulti-subunit protein, DNA encoding each subunit in the vector may bemutated in one or more positions in each subunit. When multi-subunitantibody variants are constructed, preferred sites of mutagenesiscorrespond to codons encoding amino acid residues located in thecomplementarity-determining regions (CDRs) of either the light chain,the heavy chain, or both chains. The CDRs are commonly referred to asthe hypervariable regions. Methods for mutagenizing DNA encoding eachsubunit of the protein of interest are conducted essentially asdescribed in Section V above.

[0185] 7. Preparing a Target Molecule and Binding with Phagemid.

[0186] Target proteins, such as receptors, may be isolated from naturalsources or prepared by recombinant methods by procedures known in theart. By way of illustration, glycoprotein hormone receptors may beprepared by the technique described in McFarland et al, Science 245:494-499 (1989), nonglycosylated forms expressed in E. coli are describedby Fuh et al., J. Biol. Chem 0.265: 3111-3115 (1990). Other receptorscan be prepared by standard methods.

[0187] The purified target protein may be attached to a suitable matrixsuch as agarose beads, acrylamide beads, glass beads, cellulose, variousacrylic copolymers, hydroxylalkyl methacrylate gels, polyacrylic andpolymethacrylic copolymers, nylon, neutral and ionic carriers, and thelike. Attachment of the target protein to the matrix may be accomplishedby methods described in Methods in Enzymol. 44 (1976), or by other meansknown in the art.

[0188] After attachment of the target protein to the matrix, theimmobilized target is contacted with the library of phagemid particlesunder conditions suitable for binding of at least a portion of thephagemid particles with the immobilized target. Normally, theconditions, including pH, ionic strength, temperature and the like willmimic physiological conditions.

[0189] Bound phagemid particles (“binders”) having high affinity for theimmobilized target are separated from those having a low affinity (andthus do not bind to the target) by washing. Binders may be dissociatedfrom the immobilized target by a variety of methods. These methodsinclude competitive dissociation from the immobilized target by avariety of methods. These methods include competitive dissociation usingthe wild-type ligand, altering pH and/or ionic strength, and methodsknown in the art.

[0190] Suitable host cells are infected with the binders and helperphage, and the host cells are cultured under conditions suitable foramplification of the phagemid particles. The phagemid particles are thencollected and the selection process is repeated one or more times untilbinders having the desired affinity for the target molecule areselected.

[0191] Optionally the library of phagmid particles may be sequentiallycontacted with more than one immobilized target to improve selectivityfor a particular target. For example, it is often the case that a ligandsuch as hGH has more than one natural receptor. In the case of hGH, boththe growth hormone receptor and the prolactin receptor bind the hGHligand. It may be desirable to improve the selectivity of hGH for thegrowth hormone receptor over the prolactin receptor. This can beachieved by first contacting the library of phagemid particles withimmobilized prolactin receptor, eluting those with a low affinity (i.e.lower than wild type hGH) for the prolactin receptor and then contactingthe low affinity prolactin “binders” or non-binders with the immobilizedgrowth hormone receptor, and selecting for high affinity growth hormonereceptor binders. In this case an hGH mutant having a lower affinity forthe prolactin receptor would have therapeutic utility even if theaffinity for the growth hormone receptor were somewhat lower than thatof wild type hGH. This same strategy may be employed to improveselectivity of a particular hormone or protein for its primary functionreceptor over its clearance receptor.

[0192] In another embodiment of this invention, an improved substrateamino acid sequence can be obtained. These may be useful for makingbetter “cut sites” for protein linkers, or for better proteasesubstrates/inhibitors. In this embodiment, an immobilizable molecule(e.g., hGH) receptor, biotin-avidin, or one capable of covalent linkagewith a matrix) is fused to gene III through a linker. The linker willpreferably by from 3 to 10 amino acids in length and will act as asubstrate for a protease. A phagemid will be constructed as describedabove where the DNA encoding the linker region is randomly mutated toproduce a randomized library of phagemid particles with different aminoacid sequences at the linking site. The library of phagemid particlesare then immobilized on a matrix and exposed to a desired protease.Phagemid particles having preferred or better substrate amino acidsequences in the linear region for the desired protease will be eluted,first producing an enriched pool of phagemid particles encodingpreferred linkers. These phagemid particles are then cycled several moretimes to produce an enriched pool of particles encoding consensussequence(s).

[0193] II. Generation of Antibodies.

[0194] The starting antibody may be prepared using techniques availablein the art or generating such antibodies. Exemplary methods forgenerating antibodies are described in more detail in the followingsections.

[0195] The antibody is directed against an antigen of interest.Preferably, the antigen is a biologically important polypeptide anadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated.

[0196] Where the antigen is a polypeptide, it may be a transmembranemolecule (e.g., receptor) or ligand such as a growth factor. Exemplaryantigens include molecules such as renin; growth hormone, includinghuman growth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; glucagon; clotting factors such as ProteinC; atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(tPA); bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein;such as beta-lactamase; DNase; IgE, a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factors (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5 or NT-6, or a nerve growth factor such as NGF-β, platelet-derivedgrowth factor (PDGF), fibroblast growth factors such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and —II (IGF-1 and IGF-II)des(1-3)—IGF-I (brain IGF-I), insulin-like growth factor bindingprotein; CD proteins such as CD3, CD4, CD8, CD19 and CD20;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (Ils), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; homingreceptors; adressins; regulatory proteins; integrins such as CD11a,CD11b, CD11c, CD18, and ICAM, VLA-4 and VCAM; a tumor associated antigensuch as HER2, HER3 or HER 4 receptor; and fragments of any of theabove-listed peptides.

[0197] Preferred molecular targets for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD19, CD20and CD34; members of the ErbB receptor family such as the EGF receptor,HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1,Mac12, p150,95., VLA-4, ICAM-1, VCAM and αv/β3 integrin including eitherα or β subunits thereof (e.g., anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; IgE; blood group antigens;flk2/flk3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, etc. An especially preferred target is IgE.

[0198] The antibody is raised against the antigen derived from a firstmammalian species. Preferably the first mammalian species is human.However, other mammals are contemplated such as farm, pet or zooanimals, e.g., where the antibody is intended to be used to treat suchmammals. The antigen from the first mammalian species may be isolatedfrom a natural source thereof for the purposes of generating an antibodythereagainst. However, as noted below, cells comprising the antigen canbe used as immunogens for making antibodies. In other embodiments, theantigen is produced recombinantly or made using other synthetic methods.The antibody selected will normally have a sufficiently strong bindingaffinity for the antigen. For example, the antibody may bind the antigenfrom the first mammalian species with a binding affinity (Kd) value ofno more than about 1×10⁻⁷ M, preferably no more than about 1×10⁻⁸ andmost preferably no more than about 1×10⁻⁹ M. Antibody affinities may bedetermined by saturation binding; enzyme linked immunosorbent (ELISA);and competition assays (e.g., RIAs) for example.

[0199] Also, the antibody may be subjected to other biological activityassays, e.g., in order to evaluate its effectiveness as a therapeutic.Such assays are known in the art and depend on the target antigen andintended use for the antibody. Examples include the keratinocytemonolayer adhesion assay and the mixed lymphocyte response (MFR) assayfor CD11a (each described in the Example below); tumor growth inhibitionassays (as described in WO 89/06692, for example); antibody-dependentcellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC)assays (U.S. Pat. No. 5,500,362); and agonistic activity orhematopoiesis assays (see WO 95/27062).

[0200] To screen for antibodies which bind to a particular epitope onthe antigen of interest (e.g., those which block binding of the MHM24antibody, a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. Alternatively, epitopemapping, e.g., as described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be performed to determine whether the antibodybinds an epitope of interest.

[0201] Species-dependence of the antibody is then determined. Thebinding affinity of the antibody for a homologue of the antigen used togenerate the antibody (where the homologue is from the “second mammalianspecies”) is assessed using techniques such as those described above. Inpreferred embodiments, the second mammalian species is a nonhuman mammalto which the antibody will be administered in preclinical studies.Accordingly, the second mammalian species may be a nonhuman primate,such as rhesus, cynomolgus, baboon, chimpanzee and macaque. In otherembodiments, the second mammalian species may be a rodent, cat or dog,for example. The species-dependent antibody will normally have a bindingaffinity for the antigen from the second nonhuman mammalian specieswhich is at least about 50 fold, or at least about 500 fold, or at leastabout 1000 fold, weaker than its binding affinity for the antigen fromthe first mammalian species. This binding affinity will normally be suchthat the species-dependent antibody cannot effectively be used forpreclinical studies in the second mammalian species.

[0202] While the preferred method of the instant invention fordetermining species-dependence (and for evaluating antibody mutants withimproved properties; see below) is to quantify antibody bindingaffinity, in other embodiments of the invention, one or more biologicalproperties of the species-dependent antibody and antibody mutant areevaluated in addition to, or instead of, binding affinitydeterminations. Exemplary such biological assays are described above.Such assays are particularly useful where they provide an indication asto the therapeutic effectiveness of the antibody. Normally, though notnecessarily, antibodies which show improved properties in such assays,will also have an enhanced binding affinity. Thus, in one embodiment ofthe invention where the assay of choice is a biological activity assayother than a binding affinity assay, the species-dependent antibody willnormally have a “biological activity” using “material” (e.g., antigen,cell, tissue, organ or whole animal) from the second mammalian specieswhich is at least about 50-fold, or at least about 500 fold, or at leastabout 1000 fold, less effective than its biological activity in acorresponding assay using reagents from the first mammalian species.

[0203] The species-dependent antibody is then altered so as to generatean antibody mutant which has a stronger binding affinity for the antigenfrom the second mammalian species, than the species-dependent antibody.The antibody mutant preferably has a binding affinity for the antigenfrom the nonhuman mammal which is at least about 10 fold stronger,preferably at least about 20 fold stronger, more preferably at leastabout 500 fold stronger, and sometimes at least about 100 fold or200-fold stronger, than the binding affinity of the species-dependentantibody for the antigen. The enhancement in binding affinity desired orrequired will depend on the initial binding affinity of thespecies-dependent antibody. However, the assay used is a biologicalactivity assay, the antibody mutant preferably has biological activityin the assay of choice which is at least about 100 fold better,preferably at least about 20 fold better, more preferably at least about50 fold better, and sometimes at least about 100 fold or 200 foldbetter, than the biological activity of the species-dependent antibodyin that assay.

[0204] To generate the antibody mutant, one or more amino acidalterations (e.g. substitutions are introduced in one or morealterations (e.g., substitutions) of framework region residues may beintroduced in the species-dependent antibody where the result is animprovement in the binding affinity of the antibody mutant for theantigen from the second mammalian species. Example of framework regionresidues to modify include those which non-covalently bind antigendirectly (Amit et al., Science 233: 747-753 (1986)); interactwith/effect the conformation of a CDR (Chothia et al., J Mo. Biol. 196:901-917 (1987)); and/or participate in the VL-VH interface (EP 239 400B1). In certain embodiments, modification of one or more of suchframework region residues results in an enhancement of the bindingaffinity of the antibody for the antigen from the second mammalianspecies. For example, from about one to about five framework residuesmay be altered in this embodiment of the invention. Sometimes, this maybe sufficient to yield an antibody mutant suitable for use inpreclinical trials, even where none of the hypervariable region residueshave been altered. Normally, however, the antibody mutant will compriseadditional hypervariable region alterations.

[0205] The hypervariable region residues which are altered may bechanged randomly, especially where the starting binding affinity of thespecies-dependent antibody for the antigen from the second mammalianspecies is such that such randomly produced antibody mutants can bereadily screened.

[0206] Techniques for producing antibodies, which may bespecies-dependent and therefore require modification according to thetechniques elaborated herein, follow:

[0207] A. Antibody Preparation.

[0208] (i) Antigen Preparation

[0209] Soluble antigens or fragments thereof, optionally conjugated toother molecules, can be used as immunogens for generating antibodies.For transmembrane molecules, such as receptors, fragments of these(e.g., the extracellular domain of a receptor) can be used as theimmunogen. Alternatively, cells expressing the transmembrane moleculecan be used as the immunogen. Such cells can be derived from a naturalsource (e.g., cancer cell lines) or maybe cells which have beentransformed by recombinant techniques to express the transmembranemolecule. Other antigens and forms thereof useful for preparingantibodies will be apparent to those in the art.

[0210] (ii) Polyclonal Antibodies

[0211] Polyclonal antibodies are preferably raised in non-human mammalsby multiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, thionyl chloride,or R¹N═C═CR, where R and R¹ are different alkyl groups.

[0212] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg or 5 μg of theprotein or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with ⅕ to{fraction (1/10)} the original amount of peptide or conjugate inFreund's complete adjuvant by subcutaneous injection at multiple sites.Seven to 14 days later the animals are bled and the serum is assayed forantibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the sameantigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture protein fusions. Also, aggregating agents suchas alum are suitably used to enhance the immune response.

[0213] The mammalian antibody selected will normally have a sufficientlystrong binding affinity for the antigen. For example, the antibody maybind the human anti-IgE antigen with a binding affinity (Kd) value of nomore than about 1×10⁻⁷ M, preferably no more than about 1×10⁻⁸ and mostpreferably no more than about 1×10⁻⁹ M. Antibody affinities may bedetermined by saturation binding; enzyme-linked immunosorbent assay(ELISA); and competition assays (e.g., radioimmunoassays).

[0214] To screen for human anti-IgE antibodies, a routine cross-linkingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988) can beperformed. Alternatively, epitope mapping, e.g., as described in Champe,et al. J. Biol. Chem. 270: 1388-1394 (1995), can be performed todetermine binding.

[0215] While the preferred method for determining efficacy of thepolypeptide or antibody is through quantification of antibody bindingaffinity, other embodiments envision the evaluation of one or morebiological properties of the antibody in addition to, or instead ofbinding affinity determinants. Such assays are particularly useful wherethey provide and indication as to the therapeutic effectiveness of theantibody. Normally, though not necessarily, antibodies which showimproved properties in such assays, will also have an enhanced bindingaffinity.

[0216] (iii) Monoclonal Antibodies

[0217] Monoclonal antibodies are antibodies which recognize a singleantigenic site. Their uniform specificity makes monoclonal antibodiesmuch more useful than polyclonal antibodies, which usually containantibodies that recognize a variety of different antigenic sites.

[0218] Monoclonal antibodies may be made musing the hybridoma methodfirst described by Kohler et al., Nature, 256: 495 (1975), or may bemade by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0219] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster or macaque monkey, is immunized as hereinabovedescribed to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principals and Practice, pp. 590-103 (AcademicPress, 1986)).

[0220] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phophoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), substances which prevent thegrowth of HGPRT-deficient cells.

[0221] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable form the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbar, J. Immunol. 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0222] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0223] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principals and Practice, pp. 59-103,Academic Press, 1986)). Suitable culture media for this purpose include,for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal.

[0224] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0225] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transferred intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. Recombinant production of antibodies willbe described in more detail below.

[0226] In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature 348: 552-554 (1990). Clackson etal., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., Bio/Technology 10: 779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res. 21: 2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

[0227] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851 (1984)), or bycovalently joining to the immunoglobulin polypeptide.

[0228] Typically, such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

[0229] (iv) Generation of Mutant Antibodies

[0230] Once the species-dependent antibody has been identified andisolated, it is often useful to generate a variant antibody or mutant,wherein one or more amino acid residues are altered in one or more ofthe hypervariable regions of the mammalian antibody. Alternatively, orin addition, one or more alterations (e.g. substitutions) of frameworkresidues may be introduced in the mammalian antibody where these resultin an improvement in the binding affinity of the antibody mutant forhuman IgE. Examples of framework region residues to modify include thosewhich non-covalently bind antigen directly (Amit et al. Science 233:747-753 (1986)); interact with/effect the conformation of CDR (Chothiaet al. J. Mol. Biol. 196: 901-917 (1987)); and/or participate in theVL-VH interface (EP 239 400 B1). In certain embodiments, modification ofone or more of such framework region residues results in an enhancementof the binding affinity of the antibody for the human antigen. Forexample, from about one to about five framework residues may be alteredin this embodiment of the invention. Sometimes, this may be sufficientto yield an antibody mutant suitable for use in preclinical trials, evenwhere none of the hypervariable region residues have been altered.Normally, however, the antibody mutant will comprise additionalhypervariable region alteration(s).

[0231] The hypervariable region residues which are altered may bechanged randomly, especially where the starting binding affinity of thespecies-dependent antibody is such that randomly produced antibodymutants can be readily screened.

[0232] One useful procedure for generating antibody mutants is known as“alanine scanning mutagenesis” (Cunningham, B. C. and Wells, J. A.Science 244: 1081-1085 (1989); Cunningham, B. C. and Wells, J. A. Proc.Natl. Acad. Sci. USA. 84, 6434-6437 (1991)). Here, one or more of thehypervariable region residue(s) are replaced by alanine or polyalanineresidue(s) to affect the interaction of the amino acids with the antigenfrom the second mammalian species. Those hypervariable region residue(s)demonstrating functional sensitivity to the substitutions then arerefined by introducing further or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The ala-mutants produced this way arescreened for their biological activity as described herein. Similarsubstitutions can be attempted with other amino acids, depending uponthe desired property imparted by the scanning residues.

[0233] The invention also provides a more systematic method foridentifying amino acid residues to modify. According to this method, oneidentifies hypervariable region residues in the species-dependentantibody which are involved in binding the first mammalian species andthose hypervariable region residues involved in binding a homologue ofthat antigen from the second mammalian species. To achieve this, analanine scan of the hypervariable region residues of thespecies-dependent antibody can be performed, with each ala-mutant beingtested for binding to the first and second mammalian species. Thehypervariable region residues involved in binding the antigen from thefirst mammalian species (e.g., human), and those involved in binding thehomologue of the antigen from the second mammalian species (e.g., nonhuman) are thereby identified. Preferably, those residue(s)significantly involved in binding the antigen from the second mammalianspecies, (e.g., nonhuman mammal), but not the antigen from the firstmammalian species (e.g., human), are chosen as candidates formodification. In another embodiment, those residue(s) significantlyinvolved in binding the antigen from both the first and second mammalianspecies are selected to be modified. In yet a further, but lesspreferred embodiment, those residues which are involved in binding theantigen from human IgE, but not the homologous mammalian (non-human)IgE, are selected for modification. Such modification can involvedeletion of the residue or insertion of one or more residues adjacentthe residue of interest. However, normally the modification involvessubstitution of the residue for another amino acid.

[0234] Typically, one would start with a conservative substitution suchas those shown in Table A below under the heading of “preferredsubstitutions”. If such substitutions results in a change in biologicalactivity (e.g., binding affinity), then more substantial changes,denominated “exemplary substitutions” in Table A, or as furtherdescribed below in reference to amino acid classes, are introduced andthe products screened. TABLE A Conservative Substitutions of Amino AcidResidues Preferred Original Exemplary Substi- Residue Substitutionstutions DNA Codons Ala (A) val, leu, ile Val GCA, GCC, GCG, GCU Arg (R)lys, gln, asn Lys AGA, AGG, CGA, CGC, CGG, CGU Asn (N) gln, his, lys,arg Gln AAC, AAU Asp (D) glu Glu GAC, GAU Cys (C) ser Ser UGC, UGU Gln(Q) asn Asn CAA, CAG Glu (E) asp Asp GAA, GAG Gly (G) pro, ala Ala GGA,GGC, GGG, GGU His (H) asn, gln, lys, arg Arg CAC, CAU Ile (I) leu, val,met, ala, Leu AUA, AUC, AUU phe, norleucine Leu (L) norleucine, ile,val, Ile UUA, UUG, CUA, CUC, met, ala, phe CUG, CUU Lys (K) arg, gln,asn Arg AAA, AAG Met (M) leu, phe, ile Leu AUG Phe (F) leu, val, ile,ala, tyr Leu UUC, UUU Pro (P) ala Ala CCA, CCC, CCG, CCU Ser (S) thr ThrAGC, AGU, UCA, UCC, UCG, UCU Thr (T) ser Ser ACA, ACC, ACG, ACU Trp (W)tyr, phe Tyr UGG Tyr (Y) trp, phe, thr, ser Phe UAC, UAU Val (V) ile,leu, met, phe, Leu GUA, GUC, GUG, GUU ala, norleucine

[0235] Even more substantial modifications in the antibodies' biologicalproperties are accomplished by selecting substitutions that differsignificantly in their effect on maintaining: (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation; (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu,ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp,glu; (4) basic: his, lys, arg; (5) residues that influence chainorientation: gly, pro, and (6) aromatic: trp, tyr, phe.

[0236] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class.

[0237] Nucleic acid molecules encoding amino acid sequence mutants areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette muta genesisof an earlier prepared mutant or a non-mutant version of thespecies-dependent antibody. The preferred method for making mutants issite directed mutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82:488 1985)).

[0238] In certain embodiments, the antibody mutant will only have asingle hypervariable region residue substituted, e.g., from about two toabout fifteen hypervariable region substitutions.

[0239] Ordinarily, the antibody mutant with improved biologicalproperties will have an amino acid sequence having at least 75% aminoacid sequence identity or similarity with the amino acid sequence oreither the heavy or light chain variable domain of the mammaliananti-human IgE antibody, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, and most preferably atleast 95%. Identity or similarity with respect to this sequence isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical (i.e., same residue) or similar (i.e., aminoacid residue from the same group based on common side-chain properties,supra) with the species-dependent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

[0240] Alternatively, antibody mutants can be generated by systematicmutation of the CDR regions of the heavy and light chains of theanti-IgE antibody. The preferred procedure for generating such antibodymutants involves the use of affinity maturation using phage display(Hawkins et al., J. Mol. Biol. 254: 889-896 (1992) and Lowman et al.,Biochemistry 30(45): 10832-10838(1991)). Bacteriophage coat-proteinfusions (Smith, Science 228: 1315 (1985); Scott and Smith, Science 249:386 (1990); Cwirla et al. Proc. Natl. Acad. Sci. USA 8: 309 (1990);Devlin et al. Science 249: 404 (1990); reviewed by Wells and Lowman,Curr. Opin. Struct. Biol. 2: 597 (1992); U.S. Pat. No. 5,223,409) areknown to be useful for linking the phenotype of displayed proteins orpeptides to the genotype of bacteriophage particles which encode them.The F(ab) domains of antibodies have also been displayed on phage(McCafferty et al., Nature 348: 552 (1990); Barbas et al., Proc. Natl.Acad. Sci. USA 88: 7978 (1991); Garrard et al., Biotechnol. 9: 1373(1991)).

[0241] Monovalent phage display consists of displaying a set of proteinvariants as fusions to a bacteriophage coat protein in such a way as tolimit display of the variants to only one copy per several phageparticles (Bass et al., Proteins 8: 309 (1990). Affinity maturation, orimprovement of equilibrium binding affinities of various proteins, haspreviously been achieved through successive application of mutagenesis,monovalent phage display, functional analysis, and addition of favoredmutations, as exemplified in the case of human growth hormone (Lowman &Wells, J. Mol. Biol. 234: 564-578 (1993); U.S. Pat. No. 5,534,617), aswell as the F(ab) domains of antibodies (Barbas et al., Proc. Natl.Acad. Sci. USA 91: 3809 (1994); Yang et al, J. Mol. Biol. 254: 392(1995).

[0242] Libraries of many (10⁶) protein variants, differing at definedpositions in their sequence, can be constructed on bacteriophageparticles, each of which contains DNA encoding the particular proteinvariant. After cycles of affinity purification, using an immobilizedantigen, individual bacteriophage clones are isolated, and the aminoacid sequence of their displayed protein is deduced from their DNA.

[0243] (a) Humanized and Human Antibodies

[0244] Humanization is a technique for making a chimeric antibodywherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Ahumanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom =an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al,Nature 321: 522-525 (1986); Riechman et al, Nature 332: 323-327 (1988);Verhoeyen et al., Science 239: 1534-1536 (1988)), by substituting rodentComplementarity Determining Regions (CDRs) or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Aspracticed in the present invention, the humanized IgE antibodies havesome CDR residues and possible some FR residues substituted by residuesfrom analogous sites in murine antibodies.

[0245] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al, J. Immunol. 151:2296 (1993); Chothia et al, J. Mol. Biol. 196: 901 (1987)). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992);Presta et al, J. Immunol. 151:2623 (1993)).

[0246] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences. Modelsfor particular antibody domains, for example, VH and VL domains, areconstructed separately from consensus sequences based upon F(ab)structures which have similar sequences. Three-dimensionalimmunoglobulin models are commonly available and are familiar to thoseskilled in the art. Computer programs are available which illustrate anddisplay probably three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. For example in modeling the fragment F(ab)-12 in Example 2, themurine MAE11 was used as a template for inspiration of CDR and frameworkresidues to modify in conjunction with molecular modeling to arrive atthe mutant sequence.

[0247] As another example, there can be mentioned the control antibodyMab4d5. Here, the models were constructed based upon several Fabstructures from the Brookhaven protein data bank (entries 1FB4, 2RHE,2MCP, 3FAB, IFBJ, 2HFL and 1REI). The F(ab) fragment KOL (Marquart, M.et al., J. Mol. Biol. 141: 369-391 (1980)) as first chosen as a templatefor VL and VH domains and additional structures were then superimposedupon this structure using their main chain atom coordinates (INSIGHTprogram, Biosym Technologies). Similar programs and techniques areutilized for modeling the desired antibody.

[0248] A typical analysis using molecular modeling may be conducted asfollows: The distance from the template Cα to the analogous Cα in eachof the superimposed structures is calculated for each given residueposition. Generally, if all (or nearly all) Cα-Cα distances for a givenresidue are ≦1 Å, then that position is included in the consensusstructure. In some cases the β-sheet framework residues will satisfythese criteria whereas the CDR loops may not. For each of these selectedresidues, the average coordinates for individual N, Cα, C, O and Cβatoms are calculated and then corrected for resultant deviations fromnon-standard bond geometry by 50 cycles of energy minimization using acommercially available program such as the DISCOVER program (BiosymTechnologies) with the AMBER forcefield (Weiner, S. J. et al., J. Amer.Chem. Soc. 106: 765-784 (1984)), and the Ca coordinates are fixed. Theside chains of highly conserved residues, such as the disulfide-bridgedcysteine residues, are then incorporated into the resultant consensusstructure. Next, the sequences of the particular antibody VL and VHdomains are incorporated starting with the CDR residues and using thetabulations of CDR conformations from Chothia et al. (Chothia, C. etal., Nature 342: 877-883 (1989)) as a guide. Side-chain conformationsare chosen on the basis of Fab crystal structures, rotamer libraries(Ponder, J. W. & Richards, F. M., J. Mol. Biol. 193: 775-791 (1987)) andpacking considerations. Since VH-CDR 3 may not be assignable with theabove criteria, models may be created from a search of similarly sizedloops using the INSIGHT program, derived using packing and solventexposure considerations, or created using other routine and commerciallyavailable techniques. It is preferable to subject the model to 5000cycles of energy minimization.

[0249] In this way, framework residues can be selected and combined fromthe recipient and import sequences so that the desired antibodycharacteristics, such as increased affinity for the target antigen(s),is achieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. This techniquewas used in the creation of F(ab)-12 in Example 2, where a combinationof murine CDR residues was used in conjunction with molecular modelingto create a humanized, murine anti-IgE antibody fragment.

[0250] Alternatively, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggermann et al, Year in Immunol. 7: 33(1993); and Duchosal et al., Nature 355: 258 (1992). Human antibodiescan also be derived from phage-display libraries (Hoogenboom et al., J.Mol. Biol. 227: 381 (1991); Marks et al, J. Mol Biol 222: 581-597(1991); Vaughan et al., Nature Biotech. 14: 309 (1996)).

[0251] (b) Additional Modifications

[0252] Following production of the antibody mutant, the biologicalactivity of that molecule relative to the species-dependent antibody isdetermined. As noted above, this may involve determining the bindingaffinity and/or other biological activities of the antibody. In apreferred embodiment of the invention, a panel of antibody mutants areprepared above and are screened for binding affinity for the antigenfrom the second mammalian species. One or more of the antibody mutantsselected from this initial screen are optionally subjected to one ormore further biological activity assays to confirm that the antibodymutant(s) with enhanced binding affinity are indeed useful, e.g.,preclinical studies. In preferred embodiments, the antibody mutantretains the ability to bind the antigen from the first mammalian specieswith a binding affinity similar to the species-dependent antibody. Thismay be achieved by avoiding altering hypervariable region residuesinvolved in binding the antigen from the anti-human antibody. In otherembodiments, the antibody mutant may have a significantly alteredbinding affinity from the first mammalian species (e.g. the bindingaffinity for that antigen is preferably better, but may be worse thanthe species-dependent antibody).

[0253] The antibody mutant(s) so selected may be subjected to furthermodifications, oftentimes depending upon the intended use of theantibody. Such modifications may involve further alteration of the aminoacid sequence, fusion to heterologots polypeptide(s) and/or covalentmodifications such as those elaborated below. With respect to amino acidsequence alterations, exemplary modifications are elaborated above. Forexample, any cysteines residues not involved in maintaining the properconformation of the antibody mutant also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant cross linking. Conversely, (a) cysteine bond(s) may beadded to the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment). Another typeof amino acid mutant has an altered glycosylation pattern. This may beachieved by deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that are notpresent in the antibody. Glycosylation of antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of a sugar through an etheroxygen; For example, N-acetylgalactosamine, galactose, fucose or xylosebonded to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used. Additionof glycosylation sites to the antibody is conveniently accomplished byaltering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

[0254] (v) Antibody Fragments

[0255] Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) andBrennan et al., Science 229: 81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, F(ab′)₂—SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab′) fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). (PCT patent application WO 93/16185).

[0256] (vi) Multispecific Antibodies

[0257] Multispecific antibodies have binding specificities for at leasttwo different antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin, anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin A chain,anti-CD22/anti-saporin, anti-CD7/anti-saporin, anti-CD38/anti-sporin,anti-CEA/anti-ricin A chain, anti-interferon-α(IFN-α)/anti-hybridomaidiotype, anti-CEA/anti-vinca alkaloid; BsAbs for converting enzymeactivated prodrugs such as anti-CD38/anti-alkaline phosphatase (whichcatalyzes conversion of mitomycin phosphate prodrug to mitomycinalcohol); BsAbs which can be used as fibrinolytic agents such asanti-fibrin/anti-tissue plasminogen activator (tPA),anti-fibrin/anti-urokinase-type plasminogen activator (uPA), BsAbs fortargeting immune complexes to cell surface receptors such as anti-lowdensity lipoprotein (LDL)/anti-Fc receptor (e.g. FcγRI, FcγRII orFcγRIII); BsAbs for use in therapy of infectious diseases such asanti-CD3-anti-herpes simplex virus (HSV), anti-T-cell receptor: CD3complex/anti-influenza, anti-FcγR/anti-HIV, BsAbs for tumor detection invitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA,anti-p185^(HER2)/anti-hapten; BsAbs as vaccine adjuvants; and BsAbs asdiagnostic tools such as anti-rabbit IgG/anti-ferritin, anti-horseradishperoxidase (HRP)/anti-hormone, anti-somatostatin/anti-substance P,anti-HRP/anti-FITC, anti-CEA/anti-β-galactosidase. Examples oftrispecific antibodies include anti-CD3/anti-CD4/anti-CD37,anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37. Bispecificantibodies can be prepared as full length antibodies or antibodyfragments (e.g. F(ab′)₂ bispecific antibodies).

[0258] Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light pairs, wherethe two chains have different specificities (Millstein et al., Nature305: 537-539 (1983)). Because of the random assortment of immunoglobulinheavy and light chains, these hybridomas (quadromas) produce a potentialmixture of 10 different antibody molecules, of which only one has thecorrect bispecific structure. Purification of the correct molecule,which is usually done by affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in WO 93/08829, and in Traunecker et al, EMBO J. 10: 3655-3659(1991).

[0259] According to a different approach, antibody variable domains withthe desired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2 and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportion of the three polypeptide fragments in embodiment when unequalratios of the three polypeptide chains used in the construction providethe optimum yields. It is, however, possible to insert the codingsequences for two or all three polypeptide chains in one expressionvector when the expression of at least two polypeptide chains in equalratios results in high yields or when the ratios are of no particularsignificance.

[0260] In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690. Forfurther details of generating bispecific antibodies, see for example,Suresh et al, Methods in Enzymology 121: 210 (1986).

[0261] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with large side chains (e.g., tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g., alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0262] Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0263] Techniques for generating bispecific antibodies from antibodyfragments have also been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. Brennan etal., Science 229: 81 (1985) describes a procedure wherein intactantibodies are proteolytically cleaved to generate F(ab′)₂ fragments.These fragments are reduced in the presence of the dithiol complexingagent sodium arsenite to stabilize vincinal dithiols and preventintermolecular disulfide formation. The F(ab′) fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

[0264] Recent progress has facilitated the direct recovery of Fab′-SHfragments from E. coli, which can be chemically coupled to formbispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992)describe the production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

[0265] Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc Natl. Acad.Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the VH and VL domains ofone fragment are forced to pair with the complementary VL and VH domainsof another fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruger et al.,J. Immunol. 152: 5368 (1994).

[0266] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147: 60 (1991).

[0267] (vii) Effector Function Engineering

[0268] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance the effectiveness of theantibody in binding to IgE, for example. For example, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176:1191-1195 (1992) and Shopes, B., J. Immunol. 148: 2918-2922 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

[0269] (viii) Immunoconjugates

[0270] The invention also pertains to immunoconjugates comprising theantibody described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. and enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

[0271] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII and PAP-S), momordica charantiainhibitor, curin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugate antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

[0272] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) proprionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis-p-(azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-p(diazoniumbenzoyl)-ethylenediamine),diisocyanates such as toluene 2,6-diisocyanate), and bis-active fluorinecompounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, aricin immunotoxin can be prepared as described in Vitetta et al.,Science 238: 1098 (1987). Carbon-14 labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO 94/11026.

[0273] In another embodiment, the antibody may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

[0274] (ix) Immunoliposomes

[0275] The antibody mutants disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0276] Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′; fragments of the antibody of the present invention canbe conjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989).

[0277] (x) Antibody Dependent Enzyme Mediatedprodrug Therapy (ADEPT)

[0278] The antibody of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g., a peptidyl chemotherapeutic agent, seeWO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278.

[0279] The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto convert it into its more active, cytotoxic form.

[0280] Enzymes that are useful in the method of this invention include,but are not limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxylpeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuramimidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxylacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert to prodrugs of theinvention into free active drugs (Massey, Nature 328: 457-458 (1987)).Antibody-abzyme conjugates can be prepared as described herein fordelivery of the abzyme to a tumor cell population.

[0281] The enzymes of this invention can be covalently bound to theantibody mutant by techniques well known in the art such as the use ofthe heterobifunctional crosslinking reagents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (Neuberger et al.,Nature 312: 604-608 (1984)).

[0282] (xi) Antibody-Salvage Receptor Binding Epitopefusions

[0283] In certain embodiments of the invention, it may be desirable touse an antibody fragment, rather than an intact antibody, to increasetumor penetration, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum half life.This may be achieved, for example, by incorporation of a salvagereceptor binding epitope into the antibody fragment (e.g., by mutationof the appropriate region in the antibody fragment of by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle, e.g., by DNA or peptidesynthesis).

[0284] The salvage receptor binding epitope preferably constitutes aregion wherein any one or more amino acid residues from one or two loopsof a Fc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

[0285] (xii) Other Covalent Modifications of the Antibody

[0286] Covalent modifications of the antibody are included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

[0287] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxylmethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)proprionic acid,chloroacetyl phosphate, N-alkyhnaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercura-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0288] Histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

[0289] Lysinyl and amino-terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

[0290] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0291] The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesmay be made, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵I or¹³¹I to prepare labeled proteins for use in radioimmunoassay.

[0292] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R—N═C═C—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

[0293] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

[0294] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp.79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0295] Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to: (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups such as those of cysteine; (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0296] Removal of any carbohydrate moieties present on the antibody maybe accomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem. 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties onantibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

[0297] Another type of covalent modification of the antibody compriseslinking the antibody to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxylalkylenes,in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0298] B. Vectors, Host Cells and Recombinant Methods.

[0299] The invention also provides isolated nucleic acid encoding anantibody mutant as disclosed herein, vectors and host cells comprisingthe nucleic acid, and recombinant techniques for the production of theantibody mutant.

[0300] For recombinant production of the antibody mutant, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the monoclonal antibody mutant is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody mutant). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

[0301] (i) Signal Sequence Component

[0302] The antibody mutant of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved bysignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native antibody signal sequence, thesignal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, α-factor leader (including Saccharomyces andKluyveromyces o-factor leaders), or acid phosphatase leader, the C.albicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

[0303] The DNA for such precursor region is ligated in reading frame toDNA encoding the antibody mutant.

[0304] (ii) Origin of Replication Component

[0305] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

[0306] (iii) Selection Gene Component

[0307] Expression and cloning vectors may contain a selection gene, alsotermed a selectable marker. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0308] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene produce a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin, mycophenolic acid and hygromycin.

[0309] Another example of suitable selectable markers for mammaliancells are those that enable the identification of cells competent totake up the antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, omithine decarboxylase, etc.

[0310] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium that contain methotrexate (Mtx), a competitive antagonist ofDHFR. An appropriate host cell when wild-type DHFR is employed is theChinese hamster ovary (CHO) cell line deficient in DHFR activity.

[0311] Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. (U.S. Pat. No. 4,965,199).

[0312] A suitable selection gene for use in yeast is the trpl genepresent in the yeast plasmid Yrp7 (Stinchcomb et al, Nature 282: 39(1979)). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in typtophan, for example, ATCC No.44076 or PEP4-1. Jones, Genetics 85: 12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

[0313] In addition, vectors derived from the 1.6 μm circular plasmidpKD1 can be used for transformation of Kluyveromyces yeasts.Alternatively, an expression system for large-scale production ofrecombinant calf chymosin was reported for K. lactis. Van den Berg,Bio/Technology 8: 135 (1990). Stable multi-copy expression vectors forsecretion of mature recombinant human serum albumin by industrialstrains of Kluveromyces have also been disclosed. Fleer et al.,Bio/Technology 9: 968-975 (1991).

[0314] (iv) Promoter Component

[0315] Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoa promoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

[0316] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

[0317] Examples of suitable promoter sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0318] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

[0319] Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shockpromoters—provided such promoters are compatible with the host cellsystems.

[0320] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment that also contains the SV40viral origin of replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, human β-interferon cDNA has been expressed inmouse cells under the control of a thymidine kinase promoter from herpessimplex virus. Alternatively, the rous sarcoma virus long terminalrepeat can be used as the promoter.

[0321] (v) Enhancer Element Component

[0322] Transcription of a DNA encoding the antibody of this invention byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, a-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody-encoding sequence, but is preferably located at a site 5′ fromthe promoter.

[0323] (vi) Transcription Termination Component

[0324] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

[0325] (vii) Selection and Transformation of Host Cells

[0326] Suitable host cells for cloning or expressing the DNA in thevectors herein are the prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteria such as Escherichia, e.g. E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. Subtilis and B. Licheniformis (e.g. B. Licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

[0327] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. Nidulans and A. niger.

[0328] Suitable host cells for the expression of glycosylated antibodiesare derived from multicellular organisms. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells, Luckow et al., Bio/Technology 6, 47-55 (1988); Miller etal, Genetic Engineering, Setlow et al. eds. Vol. 8, pp. 277-279 (Plenampublishing 1986); Mseda et al., Nature 315, 592-594 (1985). Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A particular cell lineof interest is insect cell line sf9. A variety of viral strains fortransfection are publicly available, e.g., the L-1 variant of Autographacalifornica NPV and the Bm-5 strain of Bombyx mori NPV, and such virusesmay be used as the virus herein according to the present invention,particularly for transfection of Spodoptera frugiperda cells. Moreover,plant cells cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

[0329] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure. See Tissue Culture, Academic Press, Kruse andPatterson, eds. (1973). Examples of useful mammalian host cell lines aremonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen. Virol. 36: 59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeycells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA,ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat livercells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68(1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2).

[0330] Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

[0331] (viii) Culturing the Host Cells

[0332] The host cells used to produce the antibody mutant of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F1O (Sigma), Minimal Essential Medium (MEM, Sigma),RPMI-1640 (Sigma), and Dulbecco

s Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing hostcells. In addition, any of the media described in Ham et al., Meth.Enzymol. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980),U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;WO 87/00195 or U.S. Pat. Re. 30,985 may be used as culture media for thehost cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as X-chlorides, where X is sodium,calcium, magnesium; and phosphates), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

[0333] (ix) Antibody Purification

[0334] When using recombinant techniques, the antibody mutant can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the antibody mutant is produced intracellularly, asa first step, the particulate debris, either host cells or lysedfragments, is removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody mutant is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

[0335] The antibody composition prepared from the cells can be purifiedusing, for example, hydroxylapatite chromatography, gel elecrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody mutant. ProteinA can be used to purify antibodies that are based on human γ1, γ2 or γ4heavy chains (Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)).Protein G is recommended for all mouse isotypes and for human y3 (Gusset al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodymutant comprises a CH3 domain, the Bakerbond ABXTM resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody mutant to be recovered.

[0336] Following any preliminary purification step(s), the mixturecomprising the antibody mutant of interest and contaminants may besubjected to low pH hydrophobic interaction chromatography using anelution buffer at a pH between about 2.5-4.5, preferably performed atlow salt concentrations (e.g., from about 0-0.25M salt).

[0337] C. Pharmaceutical Formulations.

[0338] Therapeutic formulations of the polypeptide or antibody areprepared for storage as lyophilized formulations or aqueous solutions bymixing the polypeptide having the desired degree of purity with optional“pharmaceutically-acceptable” carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”).For example, buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and other miscellaneousadditives. (See Remington's Pharmaceutical Sciences, 16th edition, A.Osol, Ed. (1980)). Such additives must be nontoxic to the recipients atthe dosages and concentrations employed.

[0339] Buffering agents help to maintain the pH in the range whichapproximates physiological conditions. They are preferably present atconcentration ranging from about 2 mM to about 50 mM. Suitable bufferingagents for use with the present invention include both organic andinorganic acids and salts thereof such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture, etc.),succinate buffers (e.g., succinic acid-monosodium succinate mixture,succinic acid-sodium hydroxide mixture, succinic acid-disodium succinatemixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartratemixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodiumhydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) and acetate buffers (e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additionally, there may be mentioned phosphate buffers, histidinebuffers and trimethylamine salts such as Tris.

[0340] Preservatives are added to retard microbial growth, and are addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0341] Isotonicifiers sometimes known as “stabilizers” are present toensure isotonicity of liquid compositions of the present invention andinclude polhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Polyhydric alcohols can be present in an amount between 0.1%to 25% by weight, preferably 1% to 5% taking into account the relativeamounts of the other ingredients.

[0342] Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (ie. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophylic polymers, such aspolyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose,glucose; disaccharides such as lactose, maltose, sucrose andtrisaccharides such as raffinose; polysaccharides such as dextran.Stabilizers are present in the range from 0.1 to 10,000 weights per partof weight active protein.

[0343] Non-ionic surfactants or detergents (also known as “wettingagents”) are present to help solubilize the therapeutic agent as well asto protect the therapeutic protein against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stressed without causing denaturation of the protein. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronice polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.). Non-ionic surfactants are present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

[0344] Additional miscellaneous excipients include bulking agents, (e.g.starch), chelating agents (e.g. EDTA), antioxidants (eg., ascorbic acid,methionine, vitamin E), and cosolvents.

[0345] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. For example, it may be desirable to further providean immunosuppressive agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0346] The active ingredients may also be entrapped in microcapsuleprepared, for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin micropheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

[0347] The formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

[0348] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semi-permeable matrices ofsolid hydrophobic polymers containing the antibody mutant, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0349] The amount of therapeutic polypeptide, antibody or fragmentthereof which will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Wherepossible, it is desireable to determine the dose-response curve and thepharmaceutical compositions of the invention first in vitro, and then inuseful animal model systems prior to testing in humans. However, basedon common knowledge of the art, a pharmaceutical composition effectivein promoting the survival of sensory neurons may provide a localtherapeutic agent concentration of between about 5 and 20 ng/ml, and,preferably, between about 10 and 20 ng/ml. In an additional specificembodiment of the invention, a pharmaceutical composition effective inpromoting the growth and survival of retinal neurons may provide a localtherapeutic agent concentration of between about 10 ng/ml and 100 ng/ml.

[0350] In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof is administered bysubcutaneous injection. Each dose may range from about 0.5 μg to about50 μg per kilogram of body weight, or more preferably, from about 3 μgto about 30 μg per kilogram body weight.

[0351] The dosing schedule for subcutaneous administration may vary fromonce a week to daily depending on a number of clinical factors,including the type of disease, severity of disease, and the subject'ssensitivity to the therapeutic agent.

[0352] D. Non-Therapeutic Uses for the Antibody Mutant.

[0353] The antibody mutants of the invention may be used as affinitypurification agents. In this process, the antibodies are immobilized ona solid phase such as Sephadex resin or filter paper, using methods wellknown in the art. The immobilized antibody mutant is contacted with asample containing the antigen to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the antigen to be purified, which is boundto the immobilized antibody mutant. Finally, the support is washed withanother suitable solvent, such as glycine buffer, pH 5.0, that willrelease the antigen from the antibody mutant.

[0354] The mutant antibodies may also be useful in diagnostic assays,e.g., for detecting expression of an antigen of interest in specificcells, tissues, or serum.

[0355] For diagnostic applications, the antibody mutant typically willbe labeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

[0356] (a) Radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. Theantibody mutant can be labeled with the radioisotope using thetechniques described in Current Protocols in Immunology, vol 1-2,Coligen et al., Ed., Wiley-Interscience, New York, Pubs. (1991) forexample and radioactivity can be measured using scintillation counting.

[0357] (b) Fluorescent labels such as rare earth chelates (europiumchelates) or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, Lissamine, phycoerythrin and Texas Red areavailable. The fluorescent labels can be conjugated to the antibodymutant using the techniques disclosed in Current Protocols inImmunology, supra, for example. Fluorescence can be quantified using afluorimeter.

[0358] (c) Various enzyme-substrate labels are available and U.S. Pat.No. 4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et a.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzym. (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

[0359] Examples of enzyme-substrate combinations include, for example:

[0360] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

[0361] (ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate aschromogenic substrate; and

[0362] (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate(e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenicsubstrate-4-methylumbelliferyl-β-D-galactosidase.

[0363] Numerous other enzyme-substrate combinations are available tothose skilled in the art. For a general review of these, see U.S. Pat.Nos. 4,275,149 and 4,318,980.

[0364] Sometimes, the label is indirectly conjugated with the antibodymutant. The skilled artisan will be aware of various techniques forachieving this. For example, the antibody mutant can be conjugated withbiotin and any of the three broad categories of labels mentioned abovecan be conjugated with avidin, or vice versa. Biotin binds selectivelyto avidin and thus, the label can be conjugated with the antibody mutantin this indirect manner. Alternatively, to achieve indirect conjugationof the label with the antibody mutant, the antibody mutant is conjugatedwith a small hapten (e.g., digloxin) and one of the different types oflabels mentioned above is conjugated with an anti-hapten antibody mutant(e.g., anti-digloxin antibody). Thus, indirect conjugation of the labelwith the antibody mutant can be achieved.

[0365] In another embodiment of the invention, the antibody mutant neednot be labeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody mutant.

[0366] The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc. 1987).

[0367] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample for binding with a limitedamount of antibody mutant. The amount of antigen in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test samples thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remainf unbound.

[0368] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, or theprotein to be detected. In a sandwich assay, the test sample to beanalyzed is bound by a first antibody which is immobilized on a solidsupport, and thereafter a second antibody binds to the test sample, thusforming an insoluble three-part complex. See e.g., U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

[0369] For immunohistochemistry, the tumor sample may be fresh or frozenor may be embedded in paraffin and fixed with a preservative such asformalin, for example.

[0370] The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody mutant is labeled with a radionucleotide (suchas ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

[0371] E. Diagnostic Kits.

[0372] As a matter of convenience, the polypeptide or antibody of thepresent invention can be provided in a kit, i.e., packaged combinationof reagents in predetermined amounts with instructions for performingthe diagnostic assay. Where the antibody mutant is labeled with anenzyme, the kit will include substrates and cofactors required by theenzyme (e.g., a substrate precursor which provides the detectablechromophore or fluorophore). In addition, other additives may beincluded such as stabilizers, buffers (e.g., a block buffer or lysisbuffer) and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will provide areagent solution having the appropriate concentration.

[0373] F. In vivo Uses for the Polypeptide or Antibody.

[0374] It is contemplated that the polypeptide or antibodies of thepresent invention may be used to treat a mammal. In one embodiment, theantibody is administered to a nonhuman mammal for the purposes ofobtaining preclinical data, for example. Exemplary nonhuman mammals tobe treated include nonhuman primates, dogs, cats, rodents and othermammals in which preclinical studies are performed. Such mammals may beestablished animal models for a disease to be treated with the antibodyor may be used to study toxicity of the antibody of interest. In each ofthese embodiments, dose escalation studies may be performed on themammal.

[0375] The antibody or polypeptide is administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantibody mutant is suitably administered by pulse infusion, particularlywith declining doses of the antibody mutant. Preferably the dosing isgiven by injections, most preferably intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

[0376] For the prevention or treatment of disease, the appropriatedosage of the antibody or polypeptide will depend on the type of diseaseto be treated, the severity and course of the disease, whether theantibody mutant is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody mutant, and the discretion of the attending physician. Theanti-human IgE antibody is suitably administered to the patient at onetime or over a series of treatments.

[0377] Depending on the type and severity of the disease, about 1 μg/kgto 150 mg/kg (e.g., 0.1-20 mg/kg) of antibody or polypeptide is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays. An exemplary dosing regimen for an anti-LFA-1 or anti-ICAM-1antibody is disclosed in WO 94/04188.

[0378] The antibody mutant composition will be formulated, dosed andadministered in a manner consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody mutant to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat a disease or disorder.The antibody mutant need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of human anti-IgE present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

[0379] G. Articles of Manufacture.

[0380] In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the antibody mutant. Thelabel on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer

s solution and dextrose solution. It may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

[0381] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0382] Example I

Preparation of Monoclonal Antibodies to IgE

[0383] Eight monoclonal antibodies (MAE10-MAE17) with the ability toblock the binding of IgE to FcεRI were prepared. Monoclonal antibodiesto IgE were prepared from the supernatants of U266B1 cells (ATCC TIB196) using affinity chromatography on an isolated anti-IgE antibody(Genentech MAE 1). For MAE12, five BALB/c female mice, age six weeks,were immunized in their foot pads with 10 μg of purified IgE in Ribi'sadjuvant. Subsequent injections were done in the same manner at one andthree weeks after the initial immunizations. Three days after the finalinjection, the inguinal and popliteal lymph nodes were removed andpooled, and a single cell suspension was made by passing the tissuethrough steel gauze. The cells were fused at a 4:1 ratio with mousemyeloma P3×63-Ag8.653 (ATCC CRL 1580) in high glucose (DMEM) containing50% w/v polyethylene glycol 4000. For MAE14, MAE15, and MAE13 theimmunizations were done in a similar manner except that for MAE13, 30 μgof IgE per injection were used and IgE fragment 315-347 (Kabat) wasassayed as a prefusion boost; For MAE 10 and MAE11, injections weregiven subcutaneously in two doses of 100 μg and a final booster of 50μg, and spleen cells were used for the fusions.

[0384] The fused cells were then plated at a density of 2×10⁵ per wellin 96 well tissue culture plates. After 24 hours HAT selective medium(hypoxanthine/aminopterin/thymidine, Sigma, #H0262) was added. Of 1440wells plated, 365 contained growing cells after HAT selection.

[0385] Fifteen days after the fusion, supernatants were tested for thepresence of antibodies specific for human IgE using an enzyme-linkedimmunosorbent assay (ELISA). The ELISA was performed as follows, withall incubations done at room temperature. Test plates (Nunc Immunoplate)were coated for 2 hours with rat anti-mouse IgG (Boehringer Mannheim,#605-500) at 1 μg/ml in 50 mM sodium carbonate buffer, pH 9.6, thenblocked with 0.5% bovine serum albumin in phosphate buffered saline(PBS) for 30 minutes, then washed four times with PBS containing 0.05%Tween 20 (PBST). Test supernatants were added and incubated two hourswith shaking, then washed four times with PBST. Human IgE (purified fromU266 cells as described above) was added at 0.5 μg/ml and incubated forone hour with shaking, then washed four times in PBST. Horseradishperoxidase conjugated goat anti-human IgE (Kirkegarrd & Perry Labs,#14-10-04, 0.5 mg/ml) was added at a 1:2500 dilution and incubated forone hour, then washed four times with PBST. The plates were developed byadding 100 μl/well of a solution containing 10 mg of o-phenylenediaminedihydrochloride (Sigma, #P8287) and 10 μl of a 30% hydrogen peroxidesolution in 25 ml phosphate citrate buffer, pH 5.0, and incubating for15 minutes. The reaction was stopped by adding 100 μl/well of 2.5 Msulfuric acid. Data was obtained by reading the plates in an automatedELISA plate reader at an absorbance of 490 nm. For MAE12, 365supernatants were tested and 100 were specific for human IgE. Similarfrequencies of IgE specificity were obtained when screening for theother antibodies. All of the monoclonal antibodies described herein wereof the IgG1 isotype except for MAE17, which was IgG2b, and MAE14, whichwas IgG2a.

[0386] Each of the IgE specific antibodies was further tested incell-based and plate assays to select for antibodies which bound to IgEin such a way as to inhibit IgE binding to FcεRI and which are notcapable of binding to FCEH-bound IgE. The results of these assays areset forth in Table 1 and Table 2 below. TABLE 1 Summary of MurineAnti-HuIgE mAb Characteristics PBL amount binding histamine blockingSchedule/ B-cell FcεRI- release² FcεRI³ mAb Immunogen Dose (μg) SourceIsotype bound IgE¹ (EC50) (EC50) MaE1 PS IgE 3 × 50 lymph IgG1 0.05μg/ml   1 μg/ml 0.3 μg node MaE10 U266 IgE   2 × 100, spleen IgG1 nobinding >100 2.5 μg 1 × 50 at 10 μg/ml     μg/ml  MaE11 U266 IgE   2 ×100, spleen IgG1 no binding >100 0.6 μg 1 × 50 at 10 μg/ml     μg/ml MaE12 U266 IgE 3 × 30 lymph IgG1 no binding >100 0.8 μg node at 10 μg/ml    μg/ml  MaE13 U266 IgE 3 × 30 lymph IgG1 no binding  >10 μg/ml  0.6μg node at 10 μg/ml MaE14 U266 IgE 5 × 50 lymph IgG2a no binding >1002.5 μg node at 10 μg/ml     μg/ml  MaE15 U266 IgE 5 × 50 lymph IgGl nobinding >100 0.6 μg node at 10 μg/ml     μg/ml  MaE16 rHIgE 5 × 1  lymphIgG1 no binding >100 0.7 μg aa315-547 node at 10 μg/ml     μg/ml  MaE17rHIgE 5 × 1  lymph IgG2b no binding >100 > 5.0 μg   aa315-547 node at 10μg/ml     μg/ml 

[0387] TABLE 2 Summary of Murine Anti-HuIgE (continued) binding toamount to block membrane IgE binding of IgE on 1 μg IgE inhibition ofin- affinity constant on U266BL FceRII (CD23) binding to vitro IgE forIgE8 mAb (EC50)⁴ IM9 (EC50)⁵ FceRII (EC50)⁶ synthesis⁷ (Kd) MaE1 0.4μg/ml   0.05 μg/ml >100 μg   (−) 5.4 × 10⁻⁸   MaE10 0.5 μg/ml   nobinding at 10 2.5 μg (−) 7 × 10⁻⁹ μg/ml MaE11 0.15 μg/ml   no binding at10 0.6 μg (+) 3 × 10⁻⁸ μg/ml MaE12  >10 μg/ml    1 μg/ml 5.0 μg (−) 4 ×10⁻⁷ MaE13 1 μg/ml no binding at 10    0.7 μg/ml (++) 5 × 10⁻⁸ μg/mlMaE14 6 μg/ml no binding at 10    2.5 μg/ml (±) 1.4 × 10⁻⁸   μg/ml MaE156 μg/ml no binding at 10    0.6 μg/ml (±) 7 × 10⁻⁸ μg/ml MaE16 10 μg/ml <0.05 μg/ml   5 μg (+) ND MaE17 10 μg/ml  no binding at 10   5 μg (++)ND μg/ml

[0388] 1. FACS based assays for analysis of murine anti-human IgEmonoclonals. Screen of murine anti-human IgE monoclonal binding to IgEon CHO 3D10 (FcεRI alpha +).

[0389] a. CHO 3D10 cells (FcεRI alpha chain stable transfectant, Hakimiet al., J. Biol. Chem. 25: 22079) at 5×10⁵ cells per sample areincubated with U266 IgE standard (lot no. 13068-46) at 10 μg/ml in 100[1 FACS buffer (0.1% BSA, 10 mM sodium azide in PBS, pH 7.4) for 30minutes at 4° C. followed by one wash with FACS buffer. The amount ofIgE binding is determined by incubating an aliquot of IgE loaded cellswith a polyclonal FITC conjugated rabbit anti-human IgG (Accurate Chem.Co. AXL-475F, lot no. 16) at 50 pg/ml for 30 minutes at 4° C. followedby three washes with FACS buffer.

[0390] b. IgE loaded cells are incubated with 100 μl of murineanti-human IgE hybridoma supernatant (murine IgG concentration rangingfrom 1 to 20 μg/ml) for 30 minutes at 4° C. followed by one wash withFACS buffer. A Genentech monoclonal anti-human IgE (MaE1) at 10 μg/ml isused as a positive control for binding. Genentech monoclonal (MAD 6P)which does not recognize IgE is used at 10 μg/ml as a negative control.

[0391] c. Monoclonal binding to human IgE on CHO cells is detected byincubating cells with 20 pg/ml FITC-conjugated, affinity purifiedF(ab′)₂ goat anti-mouse IgG (Organon Teknica, #10711-0081) for 30minutes at 4° C. followed by three washed with FACS buffer. Cells areadded to 400 μl buffer containing 2 μg/ml propidium iodide (Sigma,#P4170) to stain dead cells.

[0392] d. Cells are analyzed on a Becton Dickinson FACSCAN flowcytometer. Forward light scatter and 90 degree side scatter gates areset to analyze a homogeneous population of cells. Dead cells which stainwith propidium iodide are excluded from analysis. Hybridoma supernatantswhich do not bind IgE on CHO 3D 10 cells were considered candidates forfurther screening.

[0393] 2. Histamine release from peripheral blood basophils. Heparinizedblood was obtained from normal donors and diluted 1:4 in a modifiedTyrodes buffer (25 mM Tris, 150 mM NaCl, 10 mM CaCl₂, MgCl₂, 0.3 mg/mlHSA, pH 7.35) then incubated with 1 nM human IgE (ND) at 4° C. for 60minutes. Cells were then added to Tyrodes buffer containing either themurine monoclonal anti-IgE Abs (10 mg/ml) or a polyclonal anti-humanantiserum as the positive control, and incubated at 37° C. for 30minutes. Cells were pelleted, histamine in supernatants was acetylatedand histamine content was determined using an RIA kit (AMAC, Inc.Wesbrook, Main). Total histamine was determined from cells subjected toseveral rounds of freeze-thawing.

[0394] 3. Blocking of Fitc conjugated IgE binding to FcεRI alpha chain.The effect of the antibodies on IgE binding was studied by preincubatingFitc labelled IgE with the various MaE antibodies at 37° C. for 30minutes in PBS containing 0.1% BSA and 10 mM sodium azide pH 7.4, thenincubating the complex with 5×10⁵ 3D10 cells at 4° C. for 30 minutes.The cells were then washed three times and mean channel fluorescence at475 nM was measured. A murine anti-human IgE mAb (Mael) which does notblock IgE binding to the FcεRI alpha chain was used as a control.

[0395] 4. Analysis of murine anti-human IgE binding to membrane IgEpositive B cell U266.

[0396] a. U266 B1 cells (membrane IgE +) are cultured in base mediumsupplemented with 15% head inactivated fetal calf serum (Hyclone,#A-1111-L), penicillin, streptomycin (100 units/ml) and L-glutamine (2mM).

[0397] b. Cells (5×10⁵/aliquot) are incubated in 100 μl FACS buffercontaining murine anti-human IgE monoclonals at 10, 5, 1, 0.5 and 0.1μg/ml for 30 minutes on ice in 96 well round bottom microtiter platesfollowed by two washes with FACS buffer. The Genentech monoclonal MAE1is used as a positive control.

[0398] c. Cells are incubated in 100 μl FACS buffer containing 50 μg/ml(1:20 stock) FITC conjugated F(ab′)₂ affinity purified goat anti-mouseIgE (Organon Teknika, #1711-0084) for 30 minutes on ice followed bythree washes with FACS buffer. Cells are added to 400 μl FACS buffercontaining propidium iodide at 2 μg/ml to stain dead cells.

[0399] 5. FACS based binding assays to FcεRII (CD23)+B cell IM9.

[0400] a. IM9 human B cell myeloma (ATCC CCL 159, Ann. N.Y. Acad. Sci.190: 221-234 (1972) was maintained in GIF base medium with 10% heatinactivated fetal bovine serum, penicillin, streptomycin (100 units/ml)and L-glutamine (2 mM).

[0401] b. Cells (5×10⁵ aliquot) were incubated in 100 μl of FACS buffercontaining U266 IgE standard at 2 μg/ml for 30 minutes at 4° C. in 96well microtiter plates followed by 2 washes with FACS buffer. As acontrol, cells were incubated in buffer alone or buffer containing 2μg/ml human IgG1 (Behring Diagnostics, cat. no. 400112, lot no. 801024).

[0402] c. The cells were then incubated with murine anti-human IgEmonoclonals at 0.1 to 10 μg/ml for 30 minutes on ice. Genentechmonoclonal MAE1 was used as a positive control.

[0403] d. The cells were then incubated in 100 μl FACS buffer containingFITC conjugated F(ab′)₂ goat anti-mouse IgG at 50 μg/ml (OrganonTeknika, #1711-0084) for 30 minutes at 4° C. followed by 3 washes withFACS buffer.

[0404] e. The cells were then added to 400 μl buffer containingpropidium iodide at 2 μg/ml to stain dead cells.

[0405] f. The cells were analyzed on a Becton Dickenson FACSCAN flowcytometer. Forward light scatter and 90 degree side scatter gates wereset to analyze a homogeneous population of cells and dead cells whichstained with propidium iodide were excluded from analysis. FITC positivecells (IgE binding) were analyzed relative to cells stained with FITCrabbit anti-human IgE alone.

[0406] g. As a positive control to determine the level of CD23 on thesurface of IM9 cells in each experiment, an aliquot of cells was stainedwith Becton Dickinson murine monclonal Leu 20 (anti-CD23) at 10 μg/mlfor 30 minutes at 4° C. followed by 2 washes. The cells were thenincubated with FITC conjugated F(ab′)₂ affinity purified goatanti-murine IgG at 50 μg/ml.

[0407] 6. Antibody Blocking of FITC Conjugated IgE Binding to the LowAffinity IgE Receptor.

[0408] The binding of 40 nM FITC labelled IgE to the low affinity IgEreceptor (CD23 or FcεRI) expressed on the B lymphoblast cell IM-9 wasanalyzed by flow cytometry on a FACSCAN flow cytometer. The effect ofthe antibodies on FITC IgE binding was studied by preincubating FITC IgEwith the murine anti-human antibodies at 0.1 to 10 μg/ml chimera at 37°C. for 30 minutes in PBS containing 0.1% BSA and 10 mM sodium azide pH7.4, then incubating the complex with 5×10⁵ cells at 4° C. for 30minutes. The cells were then washed three times and mean channelfluorescence at 475 nM was measured.

[0409] 7. IgE in vitro Assay Protocol.

[0410] a. Peripheral blood mononuclear cells were separated from normaldonors.

[0411] b. Cells were washed extensively with PBS to remove as manyplatelets as possible.

[0412] c. Mononuclear cells were counted and resuspended in media at1×10⁶ cells/ml. The media was a mixture of DMEM with penicillin andstreptomycin, 15% horse serum, IL-2 (25 U/ml) and IL-4 (20 ng/ml).

[0413] d. Antibodies were added at appropriate concentrations on day 0,5 and 8.

[0414] e. Cultures were incubated in 24 well Falcon tissue cultureplates for 14 days.

[0415] f. On day 14, supernatants were removed and assayed for IgEconcentrations by an IgE specific ELISA protocol.

[0416] 8. Affinity Constant (kd) of Murine mAb for Human IgE wasDetermined by Equilibrium Binding (Scatchard) Analysis.

[0417] a. IgE (ND and PS allotypes were iodinated by the chloramine Tmethod and separated from free ¹²⁵I Na with a PD10 sephadex G25 column(Pharmacia, #17-0851-01)) in RIA buffer: PBS, 0.5% bovine serum albumin(Sigma, #A-7888), 0.05% Tween 20 (Sigma, #P-1379), 0.01% thimerosol(Sigma, #T-5125), pH 7.4. Approximately 78-95% of the post column countswere precipitated with 50% trichloroacetic acid and specific activity ofiodinated IgE preparations ranged from 1.6 to 13 μCi/μg assuming 70%counting efficiency.

[0418] b. A fixed concentration of ¹²⁵I (approximately 5×10⁴ cpm) wasadded to varying concentrations of unlabelled IgE (1 to 200 nM) in afinal volume of 0.1 ml RIA buffer in 12×75 mm polypropylene test tubes.Murine anti-human IgE mAbs (20 mM final concentration) in 0.1 ml RIAbuffer were then added for a final assay volume of 0.2 ml.

[0419] C. Samples were incubated 16-18 hours at 25° C. with continuousagitation.

[0420] d. Bound and free ¹²⁵I IgE was separated by the addition of a 0.3ml mixture of affinity purified goat anti-mouse IgG (BoehringerMannheim, #605-208) coupled to CNBr activated Sepharose 4B (Pharmacia,#17-0430-01) and carrier protein A sepharose (Repligen, #IPA 300) in RIAbuffer and incubated 1 to 2 hours at 25° C. with continuous agitation.RIA buffer (1 ml) was then added, and tubes were centrifuged 5 minutesat 400× g. Samples were then counted to determine total counts. Thesupernatants were aspirated with a finely drawn pasteur pipet, sampleswere recounted and bound versus free counts were calculated.

[0421] e. Scatchard analysis was performed utilizing a Fortran program(scanplot) based on the Ligand program written by P. Munson at NIH.Scatplot uses a mass action equation fitting bound as a function oftotal using the Rodbard type regression analysis.

Example 2 Preparation of Humanized MaE11 Introduction

[0422] The following example describes various preparations of humanizedMaE11 wherein residues were modified via site-directed mutagenesis toarrive at 12 anti-IgE MaE11 variants [F(ab)1-12]. The residues ofF(ab)12 were used as the template to create rhuMaE25 or E25, a highlypotent anti-IgE antibody described in Application PCT/US92/06860, filedAug. 14, 1992.

Methods

[0423] The murine anti-human IgE mAb MaE11, shown in FIG. 1, wasmodified by site-directed mutagenesis (Kunkel, T. A. (1985), Proc. Natl.Acad. Sci. USA 82: 488) from a deoxyuridine-containing templatecontaining a human k-subgroup I light chain and human subgroup III heavychain (VH-CH1) in a pUC119-based plasmid, pAK2 (Carter et al. (1992),Proc. Natl. Aca Sci. USA 89: 4285) to obtain the variant F(ab)-1.F(ab)-2 was constructed from the F(ab)-1 template and all otherhumanized F(ab) variants were constructed from a template of F(ab)-2.The plasmids were transformed into E. coli strain JM101 for thepreparation of double- and single stranded DNA (Messing, J. (1979),Recomb. DNA Tech. Bull. 2: 43; Ausubel et al., Current Protocols inMolecular Biology, Unit 1 (1997)). Both the light and heavy chainresidues were completely sequenced using the dideoxynucleotide method.The DNA encoding light and heavy chains was then subcloned into aderivative of the E. coli F(ab) expression plasmid, pAK19 (Carter et al.(1992), Biotechnology 10: 163). The derivative lacked the hinge cyteinesthat form the interheavy chain disulfides in F(ab′)₂ fragments. TheF(ab) fragments, as opposed to full-length IgG antibodies, facilitatedthe analysis of a moderately large number of variants because E. coliexpression could be used rather than mammalian cell culture techniques.These individual variants are described in application WO 93/04173published Mar. 4, 1993. Once the best variant was determined, it wassubsequently subcloned into a plasmid encoding a full-length human IgG1(see below).

[0424] The expression plasmids were transformed into E. coli strainMM294 (Meselon, M and R. Yuan (1968), Nature 217: 1110), and a singlecolony was grown in 5 ml of 2YT media-carbenicillin (100 μg/ml) for 5-8hours at 37° C. The culture (5 ml) was then added to 100 ml of AP5media-carbenicillin (100 μg/ml) and allowed to grow for 16 hours in a500 ml shaker flask at 37° C. The culture was centrifuged at 4,000× gand the supernatant removed. After freezing for 1 hour, the pellet wasresuspended in 5 ml cold 10 mM Tris, 1 mM EDTA and 50 μl 0.1 Mbenzamidine (Sigma, St. Louis), the latter of which was added to inhibitproteolysis. After gentle shaking on ice for 1 hour, the sample wascentrifuged at 10,000× g for 15 minutes. The supernatant was applied toa protein A-Sepharose CL-4B (Pharmacia) column (0.5 ml bed volume) thenwashed with a 10 ml solution of 3 M potassium chloride/100 mL Tris, pH8.0, followed by elution with 100 mM acetic acid (2.5 ml), pH 2.8 into 1M Tris, pH 8.0 (0.5 ml).

[0425] The F(ab) buffer was then exchanged into PBS using a Centricon-30(Amicon) and concentrated to a final volume of 0.5 ml. SDS-PAGE gels ofeach F(ab) fragments were run in order to ascertain purity. The F(ab)concentrations were determined by using a 0.1% ε₂₈₀ of 1.0. Theextinction coefficient was determined by using the concentration ofprotein from an amino acid analysis of purified F(ab)-2 and the A₂₈₀ forthis same sample.

[0426] Selected F(ab) fragments were analyzed directly by liquidchromatography/mass spectrometry to confirm their molecular weight.Samples were injected into a packed capillary liquid chromatographysystem (Henzel, W. J. et al. (1990), Anal Biochem. 187: 228) andanalyzed directly with a Sciex API 3 mass spectrometer. The highercharge states of human growth hormone (m.w. 22,256.2), acquired usingthe same instrument parameters as those used for the samples, were usedfor calibration.

[0427] For generation of full-length human IgG1 versions of humanizedMaE11, the heavy and light chains were subcloned separately intopreviously described pRK plasmids (Gorman, C. M. et al. (1990), DNAProtein Eng. Tech. 2: 3). Appropriate heavy and light chain plasmids(depending upon the sequence change(s) desired) were cotransfected intoan adenovirus-transformed human embryonic kidney cell line, known as 293(Graham, F. L. et al. (1977), J. Gen. Virol. 36: 59), using a highefficiency procedure (Graham et al., supra & Gorman, C. M., Science 221:551). Media was changed to serum free and harvested daily for up to 5days. Antibodies were purified from the pooled supernatants usingprotein A-Sepharose CL-4B (Pharmacia). The eluted antibody was bufferexchanged into PBS by G25 gel filtration, concentrated byultrafiltration using a Centriprep-30 or Centricon-100 (Millipore), andstored at 4° C. The concentration of antibody was determined using totalIgG-binding ELISA. The results are described in Table 4.

[0428] Soluble Receptor Assay:

[0429] A 96-well assay plate (Nunc) was coated with 0.05 ml of the FcεRIα-chain IgG chimeric receptor in coating buffer (50 mM carbonate,bicarbonate, pH 9.6) for 12 hours at 4-8° C. The wells were aspiratedand 250 μl blocking buffer (PBS, 1% BSA, pH 7.2) was added and incubatedat 1 hour at 4° C. In a separate assay plate the samples and referencemurine MaE11 were titered from 200 to 0.001 μg/ml by 1:4 dilutions withassay buffer (0.5% BSA and 0.05% Tween 20, PBS, pH 7.2) and an equalvolume of 10 ng/ml biotinylated IgE (O-Shannessy, D. J. et al. (1984),Immunol. Let. 8: 273) was added followed by incubation of the plate for2-3 hours at 25° C. The FcεRI-coated wells were washed three times withPBS and 0.05% Tween 20 (Sigma) and 50 μl from the sample wells weretransferred and incubated with agitation for 30 minutes at 25° C. Asolution of Streptavidin-HRP (500 μg/ml, Sigma), diluted to 1:5000 inassay buffer, was added at 50 μl/well followed by incubation of theplate for 15 minutes with agitation, and washing as describedpreviously. Fifty μl/well of Microwell Peroxidase Substrate (Kirkgaard &Perry Laboratories) was added and the color was developed for 30minutes. The reaction was stopped by adding an equal volume of 1 N HCl,and the absorbance measured at 450 μm. The concentration at 50%inhibition was calculated by plotting percentage of inhibition versusconcentration of blocking antibody with a nonlinear four-parameter curvefit using the Kaleidagraph data analysis application (Synergy Software).The results are reported in Table 3.

[0430] FACS-Based Binding Assays:

[0431] The ability of the antibody to inhibit FITC-conjugated (Goding,J. W. (1976), J. Immunol. Methods 13: 215) IgE binding to the α-chain ofthe high-affinity FcεRI receptor expressed on CHO 3D10 cells (Hakimi, J.et al. (1990), J. Biol. Chem. 265: 22079) was determined by flowcytometry. FITC-conjugated IgE (40 nM) was preincubated with theantibody (0.3-1.0×10⁻⁶ M) at 37° C. for 30 minutes in FACS buffer (PBS,0.1% BSA, and 10 mM sodium azide, pH 7.4). The complex was thenincubated wth 5×10⁵ CHO CD10 cells at 4° C. for 30 minutes. The cellswere washed three times with FACS buffer and mean channel fluorescenceat 475 nm measured on an FACScan flow cytometer (Becton Dickinson).MaE1, a murine anti-human IgE mAb that does not block IgE binding to theFcεRI α-chain, was used as a positive control and MOPC21 (Cappel), amurine monoclonal that does not recognize IgE, was used as a negativecontrol. The results are described in FIG. 3.

[0432] Binding of Antibodies to IgE-Loaded FcεRI:

[0433] Antibody binding to human IgE associated with the a-subunit ofFcεRI expressed on CHO 3D10 cells with 10 μg/ml human IgE for 30 minutesat 4° C. Cells were washed three times followed by a 30 minuteincubation with varying concentrations of either murine anti-human IgEmAbs MaE1 or MaE11 or the humanized mAb variant 12 [F(ab)12]. MOPC21(murine IgG1) was used as a control for the murine mAbs, whereashumanized 4D5 mAb (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285(1992), human IgG1) was used as a control for F(ab)12. Binding of murinemAbs was detected with a FITC-conjugated F(ab′)₂ goat anti-mouse IgE (10μg/ml). Humanized mAb binding was detected with a FITC-conjugatedF(ab′)₂ goat anti-human IgG (50 μg/ml), which had been affinity purifiedon an IgE column to minimize cross-reactivity to IgE. The results aredescribed in FIG. 4.

[0434] Computer Graphics Models of Murine and Humanized F(ab)s:

[0435] The sequences of F(ab) VL and VH domains of FIG. 1 were used toconstruct a computer graphics model of the murine MaE11 VL-VH domains.This model was subsequently used to determine which framework residuesshould be incorporated into the humanized antibody which resulted in thecreation of F(ab)-2. Models of the humanized variants were alsoconstructed to verify correct selection of murine framework residues.Construction of the models was performed as described in Carter et al.(1992), Proc. Natl. Acad. Sci. USA 89: 4285; Eigenbrot, C. et al.(1993), J. Mol. Biol. 229: 969.

Results

[0436] Design of Humanized MaE11 Antibodies:

[0437] The present study of humanized antibodies used a human consensussequence. This is in contrast to other humanization techniques that haveused human sequences closest to the murine Ig of interest. Shearman, C.W. et al. (1991), J. Immunol. 147: 4366; Kettleborough, C. A. et al.(1991), Protein Eng. 4: 773; Tempest, P. R. et al. (1991), Biotechnology9: 266; Co, M. S. et al. (1991), Proc. Natl. Acad. Sci. USA 88: 2869;Routledge, E. G. (1991), Eur. J. Immunol. 21: 2717. This human consensussequence consisted of a framework based on human VH subgroup III and VLκsubgroup I as defined in Kabat et al. (1991), Sequences of proteins ofImmunological Interest, 5 ed., National Institute of Health, Bethesda,Md. F(ab)-1 was created by grafting the six CDR's, as defined by Kabat,supra, onto a human IgG1 framework. All framework residues were retainedas human. This variant would best be described as a straight “CDR swap.”F(ab)-1 showed no detectable, inhibition of IgE binding to the receptor(Table 3). The failure of such “CDR swap” variants to bind theirantigens has been reported previously. Carter et al., supra; Tempest etal., supra. Note that the exact sequence of F(ab)-1 is not described inTable 3, however, this sequence can be inferred by substituted MaE11murine Kabat CDR residues (indicated in brackets) for correspondinghuman residues. FIG. 1 indicates Kabat CDRs by right-hand and left-handbrackets, while Chothia CDRs are indicated by underline.

[0438] In order to assist in interpretation and reduce confusion, humanresidues are written in normal type, while murine residues appear initalics. Where residue substitutions are indicated, the first residue isthe one being replaced, the second the one being inserted, and thenumber the Kabat designation of the native sequence.

[0439] The F(ab)-2 variant was based on molecular modeling. In thismolecule, several murine framework residues were incorporated into thehuman framework. In F(ab)-2, the definition of CDR's provided by Kabatet al., supra (which are based on interspecial sequence variability)were used except for CDR-H1 and CDR-H2.

[0440] CDR-H1 definitions based on sequence variability (Kabat et al.,supra) between one based on crystallographic studies of antigen-antibodycomplexes (Chothia, C. et al. (1989), Nature 342: 877) differsignificantly (FIG. 1). Therefore, CDR-H1 was redefined to include bothdefinitions, i.e., human residues 26-35.

[0441] The definition of CDR-H2 based on sequence variability (Kabat etal) contains more residues than the one based on antibody-antigencrystal structures (Chothia et al.) [see FIG. 1: Kabat CDR's are definedby brackets, Chothia by underline]. Because no crystal structure wasdiscovered which indicated antibody-antigen contacts for antibody humanresidues 60-65, CDR-H2 was modified to include a hybrid of bothdefinitions, i.e., human residues 50-58. As a result, in F(ab)-2 ashorter version of CDR-H2 was used as compared with F(ab)-1.

[0442] As a result, F(ab)-2 was created with the minimal amount ofchanges from human to murine residues which were believed to be requiredfor maintenance of binding. An additional 10 variants were created inorder to test the effects of buried residues on CDR conformations, aswell as to evaluate the predictive effects of molecular modeling ofsignificant framework residues and examine other interesting residues.

[0443] To test the effects of buried residues on CDR conformation,F(ab)-3 to F(ab)-7 were constructed in which murine residues werechanged back to human ones. As is indicated in Table 4 (by F(ab)-3 andF(ab)-4), the side chains at VL4 and VL33 have minimal effect on bindingand presumably the conformation of CDR-L1 in the humanized antibody.

[0444] Modeling suggested that framework residue VH 24 might affect theCDR-L1 conformation and VH 37 could affect the VL-VH interface. However,substitution of the human residue into at VH 24 [F(ab)-5] or VH37[F(ab)-7] showed minimal reduction in binding. In contract, replacementof the murine Phe at framework position VH 78 with human Leu [F(ab)-6]caused a significant reduction in binding. The models suggest that thisresidue is influencing the conformation of CDR-H1 and/or CDR-H2.

[0445] F(ab)-9 to F(ab)-12 examined the replacement of human residueswith murine. All four of these variants exhibited substantialimprovement in binding compared with F(ab)-2 (See Tables 3, 4 and FIG.3). In F(ab)-9, which exhibited five-fold improved binding over F(ab)-2,two residues in CDR-H2 (as defined by Kabat et al., supra) were changedto murine residues: Ala VH 60 Asn and Asp H61 Pro. The Pro substitutioncould have altered the CDR-H2 conformation and/or rigidity and Asn H60is anticipated to be buried at the VL-VH interface, possible interactingwith Asp VL1.

[0446] F(ab)-10, which displayed substantially improved binding relativeto F(ab)-2, was a variant in which all buried residues (defined asresidues with accessible surface area being less than 5% of that of thefree amino acid) in both the VL and VH domains were those of murineMaE11. In essence, F(ab)-10 can be thought of as murine MaE11 in whichonly exposed, non-CDR residues in VL and VH were changed to humanresidues.

[0447] To determine whether the improved binding of F(ab)-10 was due toone or a few residues, variants F(ab)-11 and F(ab)-12 were created.Instead of F(ab)-2, F(ab)-9 was used as the base template from which toprepare these variants because it exhibited a fivefold improved binding.Modelling suggested that sidechains at VH 63 and VH67 could affect theconformation of CDR-H2. VH 63 is considered part of CHR-H2 as defined byKabat et al., supra, but not as defined by Chothia et al., supra. VH 67is considered a framework residue in both definitions. In F(ab)-11, VH63 and VH 67 were the murine residues Leu and Ile, respectively. InF(ab)-12, only VH 67 was changed to murine Ile.

[0448] In both the soluble receptor (Table 4) and cell based assays(Table 4, FIG. 3), both of the variants F(ab)-11 and F(ab)-12 exhibitedbinding that was at least as good as F(ab)-10, and better than F(ab)-9.This suggests that the improved binding of F(ab)-10 was not due torepacking of the VH domain interior with murine residues, but was due tothe effect of only a single residue, i.e. VH 67.

[0449] F(ab)-8 was constructed replacing human VL 55 residue Glu withmurine Gly as well as well as similar substitutions at VL 57 of Gly forGlu. F(ab)-2 used the human residues, while F(ab)-8 substituted themurine residues at these positions. As can be quickly surmised fromTable 3, the substitution of these residues had no impact upon receptorbinding. TABLE 3 Changes from F(ab)-2^((a)) Variant VL VII Concentrationat 50% inh. (ng/ml) Mean, std. dev.^((b))$\frac{{F({ab})}\text{-}X}{{F({ab})}\text{-}2}$

$\frac{{F({ab})}\text{-}X}{MaE11}$

F(ab)-1 Leu 4 Met Val 24 Ala >100,000 >16.0^((c)) >560 Arg 24 Lys Ile 37Val Glu 55 Gly Thr 57 Ser Gly 57 Glu Ala 60 Asn Val 63 Leu Gly 65 AsnPhe 78 Leu F(ab)-2 — — 6083, 1279 1.0 34 F(ab)-3 Leu 4 Met — 9439, 5081.6 53 Met 33 Leu F(ab)-4 Leu 4 Met — 6770, 349 1.1 3.8 F(ab)-5 — Val 24Ala 9387, 733 1.6 52 F(ab)-6 — Phe 78 Leu 17,537, 4372 2.9 24 F(ab)-7 —Ile 37 Val 8622, 107 1.4 48 F(ab)-8 Glu 55 Gly — 5799, 523 1.0 32 Gly 57Glu F(ab)-9 — Ala 60 Asn 1224, 102 0.20 6.8 Asp 61 Pro F(ab)-10 Ala 13Val Val 48 Met 842, 130 0.14 4.7 Val 19 Ala Ala 49 Gly Val 58 Ile Ala 60Asn Leu 78 Val Vat 63 Leu Val 104 Leu Phe 67 Ile Ile 69 Val Met 82 LeuLeu 82c Ala F(ab)-11 — Ala 60 Asn 416, 66 0.07 2.3 Asp 61 Pro Val 63 LeuPhe 67 Ile F(ab)-12 — Ala 60 Asn 501, 84 0.08 2.8 Asp 61 Pro Phe 67 IleMaE11 — — 179, 63 0.03 1.0

[0450] The F(ab) variants determined to have binding closest to murineMaE11, namely F(ab)-2, F(ab)-9, F(ab)-10 and F(ab)-12 were used togenerate full-length IgG1 molecules. The binding of these moleculesrelative to variant F(ab)-2 or MaE11 was comparable to the bindingexhibited by the F(ab) fragments. These results are reported in Table 4.TABLE 4 Humanized MaE11 IgG1 variants Concentration at 50% inh. (ng/ml)Variant X Variant X Full length variant mean, std. dev.^((a))IgG1-2^((b)) MaE11 IgG1-2 7569, 1042 1.0 16.9 IgG1-9 3493, 1264 0.46 7.8IgG1-10 1118, 172 0.15 2.5 IgG1-12 1449, 226 0.19 3.2 MaE11  449, 530.06 1.0

[0451] Binding of MeE11 to IgE-Loaded FcεRI:

[0452] Murine MaE11 prevents binding of free IgE to FcεRI on mast cellsbut does not trigger histamine release by binding to IgE-loaded FcεRI.As shown in FIG. 4, both murine MaE11 and humanized variant 12 (IgG1-12)as well as the negative isotype control antibody MOPC21 and the negativeisotype control humanized 4D5 (Carter et al., supra) did not bindIgE-loaded FcεRI on CHO 3D10 cells. In contrast, the murine MaE1antibody, which binds to IgE but does not prevent IgE binding to FcεRI,bound to the IgE-loaded FcεRI. Unlike the human IgG1 control (humanized4D5), the murine IgG1 isotype (as represented by MOPC21) exhibits anonspecific background binding of approximately 10% on these cells.MaE11 did not give staining above the MOPC21 control and humanizedvariant 12 did not give staining above the humanized 4D5 control (FIG.4).

[0453] Partial Alanine Scanning of CDR Residues Important in IgEBinding:

[0454] The sequences of the MaE11 CDR's indicate a preponderance ofcharged residues (FIG. 1). CDR-L1 contains three Asp residues, whereasCDR-L3 possesses H is, Glu and Asp. CDR-H3 has three H is residues. Themodels of murine and humanized MaE11 illustrated the spatial proximityof the all of these charged residues (not shown). In contrast, the loneAsp 54 in CDR-H2 is spatially separated from the other charged residues.Alanine was substituted, by site-directed mutagenesis (Kunkel, T. A.(1985), Proc. Natl. Acad. Sci. USA 82: 488), for each of these chargedresidues to generate variants. In CDR-L 1, alteration of one of thethree Asp residues, Asp VL32b, effectively abolished IgE binding[F(ab)-16; Table 5], whereas substitution of the other Asp residues hadminimal effect [F(ab)-14; F(ab)-15]. Simultaneous alteration of Glu VL93and Asp VL94 to alanine in CDR-L3 [Fa(ab)-17; Table 5], also reducedbinding, although not to the same extent as did replacement at VL32b.Individual substitution of the three H is residues in CDR-H3 with Alaresulted in either slightly improved binding [F(ab)-21] or a three-foldreduction in binding [F(ab)-20 and F(ab)-22]. However, simultaneousalteration of all three H is residues abolished binding [F(ab)-19].Although it is not readily determinable whether the charged residues areinvolved in direct binding to IgE or to provide some conformationalstability to their respective CDR's, variants F(ab)-13 to F(ab)-22 showthat CDR-L1 and CDR-H3 are important determinants in IgE binding. TABLE5 Humanized Mae11 F(ab) CDR Residue Variants Concentration at Changesfrom F(ab)-2^((a)) 50% inh. (ng/ml) F(ab)-X Variant VL VH mean, std.dev.^((b)) F(ab)-2 F(ab)-2 — — 6083, 1279 1.0 F(ab)-13 Asp 30 Ala— >100,000 >16.0^((c)) Asp 32 Ala Asp 32b Ala F(ab)-14 Asp 30 Ala —3452, 183 0.57 F(ab)-15 Asp 32 Ala — 6384, 367 1.0 F(ab)-16 Asp 32b— >100,000 >16.0 Ala F(ab)-17 Glu 93 Ala — 17,456, 7115 2.9 Asp 94 AlaF(ab)-18 — Asp 54 Ala 2066, 174 0.34 F(ab)-19 — His 97Ala >100,000 >16.0 His 100a Ala His 100c Ala F(ab)-20 — His 97 Ala19,427, 8360 3.2 F(ab)-21 — His 100a 2713, 174 0.45 Ala F(ab)-22 — His100c 15,846, 8128 2.6 Ala

Summary and Conclusion

[0455] The creation of a functional, humanized murine anti-IgE antibodyfrom MaE11 involves the substitution of several murine frameworkresidues into the human framework. In addition, mapping of the chargedCDR residues indicated that some of these are important in theantibody-IgE interaction.

[0456] In agreement with previous studies (Carter et al., supra;Shearman, C. W. et al. (1991), J. Immunol. 147: 4366; Kettleborough, C.A. et al. (1991), Protein Eng. 4: 773; Tempest, P. R. (1991),Biotechnology 9: 266), variants F(ab)-1 to F(ab)-12 indicate thatframework residues can have a significant effect on antibody function.This is particularly emphasized when considering F(ab)-1, which is astraight CDR swap in which only the six murine CDR's were transplantedonto the human framework residues. A potential explanation for thisinvolves CDR-H2. The buried hydrophobic residues at positions VH63 andVH67 could affect the conformation of CDR-H2. Variants were createdcontaining four combinations at positions VH63 and VH67, i.e., murineLeu and Ile, respectively [MaE11 and F(ab)-11], Val and Phe [F(ab)-2],Leu and Phe [F(ab)-1], and Val and Ile [F(ab)-12]. The clear inferencefrom the binding data of these four variants indicates that theimportant residue is VH67, which must be the murine Ile in order toprovide affinity comparable to murine MaE11. In F(ab)-1, this residuewas the human Phe.

[0457] Of the 12 residues in F(ab)-1 retained as human [compared withF(ab)-2], 8 were separately changed to murine in other variants. Threechanges had no effect on binding: VL4 [F(ab)-4]; VL55 and VL 57[F(ab)-8]. Two residue substitutions: VH60 and VH 61 [F(ab)-9], improvedbinding, whereas three reduced binding: VH24 [F(ab)-5]; VH37 [F(ab)-7]and VH78 [F(ab)-6].

[0458] The variant F(ab)-10 was designed with the hypothesis suggestedby Padlan (Padlan, E. A. (1991), Mol Immunol. 28: 489), who proposedthat murine antibody immunogenicity can be reduced by changing onlyexposed framework residues. In this variant, the hydrophobic interior ofboth the VL and VH domains, in other words, the variant was the murineMaE11 in which only exposed framework residues in VL and VH were changedto the human sequence. Although F(ab)-10 exhibited binding close to thatof the murine MaE11, a change in a single amino acid domain, VH67 fromhuman to murine effected the same improvement in binding [F(ab)-12,IgG1-12].

[0459] The humanized variant exhibiting binding comparable to murineMaE11, which also required the fewest changes, was F(ab)-12. Thisvariant replaced only 5 human framework residues with murine (VL4, VH24,VH37, VH67 and VH78. Four of these residues were determined by molecularmodeling. The fifth, VH67, as well as the CDR-H2 residues VH60 and VH61,were included by using the molecular models in an effort to improve thebinding of the initial variant F(ab)-2.

Example 3 Histamine Release Assay Introduction

[0460] This is a rat mast cell histamine assay (RMCHA) which measuresquantitatively the biological activity of a recombinant humanized,monoclonal anti-IgE antibody based on the ability of the antibody toblock histamine release from allergen-sensitized RBL 48 cells.Furthermore, this determination is made under physiological conditionssimilar to those of the human body. The RBL 48 cell line was derivedfrom the parental rat mast cell line RBL 2H3 which has been subsequentlytransfected with the α-subunit of the high affinity human IgE receptor(FcεR1). Gilfillan A. M. et al., J. Immunol. 149(7): 2445-2451 (1992).

Methods

[0461] RBL 48 cells (Gilfillan et al., supra) are grown in sIMDM,Iscove's modified Dulbecco's media supplemented with 10% fetal calfserum, 2 mM glutamine, and 500 μg/ml of active geneticin (Gibco,#860-1811) in a T175 tissue culture flask (Falcon #3028) at 37° C. in ahumidified 5% CO₂ incubator (Fischer, model #610). The cells wereharvested by exposure to 4 mL solution of PBS/0.05% trypsin/0.53 mM EDTAfor 2 minutes at 37° C. followed by centrifugation (400× g, 10 min.) andresuspension in fresh sIMDM. The cells in suspension were counted with ahemocytometer (Reichert-Jung) and the density was adjusted to 0.4×10⁶cells/ml. The cells were then seeded at 100 μl/well (40,000 cells perwell) in the inner 60 wells of a 96-well, U-shaped tissue culture plate(Linbro) and cultured for 24 hours at 37° C. in the humidified 5% CO₂incubator. After being washed once with 200 μl/well of sIMDM (viaaspiration), the cells were preincubated for 30 minutes with 90 μl/wellof a solution of assay diluent (sIMDM, 3 U/ml Na-heparin) withragweed-specific IgE (RSIgE, 10 ng/ml, 23.48 ng/ml total IgE, 1.43%ragweed-specific human plasma, North American Biological, lot#42-365054).

[0462] After the preincubation period, 10 μl/well of either anti-IgEantibody (diluted in assay diluent, 0.06-39.4 μg/ml) or assay diluent(for total histamine release, background, and ragweed controls) wereadded to the cells, and the plate was incubated for 24 hours in 5% CO₂at 37° C. in the incubator After the incubation, the cells wereaspirated and washed 3× with 200 μl/well sIMDM. Following the washing,the cells were incubated with 100 μl/well of either (1) 0.5% tritonsolution (for total histamine release), (2) histamine release buffer(HRB, 50% D₂O, 0.8% NaCl, 1.3 mM CaCl₂, sIMDM, or (3) ragweed antigen(NIH #A-601-903A-185, 0.1 μg/ml in HRB) at 37° C. for 30 minutes and thereaction was stopped by placement on ice. (100% D₂O=100% D₂O, 0.8% NaCl,1.3 mM CaCl₂).

[0463] The plate was centrifuged for 5 minutes at 900× g (2460 rpm) at4° C., and the supernatants were harvested and diluted {fraction (1/80)}in PBS ({fraction (1/1000)} in PBS for total histamine release control)for histamine determination using the Histamine Enzyme Immunoassay Kit(Immunotech #1153). The supernatants (100 μl/well) were transferred toacylation tubes containing acylation powder (per kit) and-reacted with50 μl acylation buffer (per kit) for 30 minutes at ambient temperature.The acylated histamine (50 μl/well) was then transferred to aconjugation plate (per kit) and incubated with 200 μl/well ofhistamine-acetylcholinesterase conjugate (per kit) for 18 hours at 4° C.

[0464] After this incubation, the wells were blotted and rinsed toremove unbound conjugate by washing 4× with 300 μl/well of washingbuffer (Immunotech kit, #1153). The chromatogenic substrate(acetylthiocholine, dithionitrobenzoate, 200 μl/well, per kit) was addedand incubated in the dark at ambient temperature for 30 minutes. Thereaction was stopped by the addition of stop solution (50 μl/well, perkit) and the absorbance at 405 nm with a 620 nm reference was determinedon a SLT 340 ATTC plate reader. The intensity of absorbance is inverselyproportional to the histamine concentration (expressed as nM) which isdetermined from the histamine standard curve (from the enzymeimmunoassay kit, AMAC). The percent total histamine release wascalculated from data of histamine concentration and the percentinhibition was calculated by 100%-total histamine release. The resultsare indicated in FIG. 5.

Summary and Conclusion

[0465] The graph of molar ratio anti-IgE vs. percent inhibition ofragweed-induced histamine release indicates that the F(ab) form of E26antibody has superior ragweed-induced histamine release properties thanthe F(ab) form of E25 antibody. E26 inhibits ragweed-induced histaminerelease in a dose dependent manner with a half-maximal inhibition molarratio of 44:1 (anti-IgE:RSIgE). In contrast, E25 only inhibitsragweed-induced histamine release at a very high molar ratio (between200:1 to 1550:1 anti-IgE:RSIgE). The half-maximal inhibition molar ratiofor the E25 curve could be estimated to be between 400:1 to 500:1.Therefore, based on the half-maximal inhibition molar ratio data, whichis a measure of the binding affinity of a molecule, the E26 moleculebinds to RSIgE approximately 10-times better than the E25 molecule.

Example 4 Phage Display Example Introduction

[0466] This example describes specific affinity-improved anti-IgEantibodies generated through monovalent phage display and selection ofF(ab) fragments derived from the E25 humanized anti-IgE antibody (Prestaet al, J. Immunol. 151:2623 (1993).

Methods

[0467] I. Construction of Monovalent F(ab)-Phage Libraries.

[0468] Several F(ab) libraries were constructed. As a starting vector,an E25 variant containing the VL substitution D32E (to eliminate IsoAspisomerization) was fused to the C-terminal domain of bacteriophageM13g3p by known techniques, see for example Bass et al., Proteins 8: 309(1990). This plasmid, which was known as p426 appears in FIG. 10. First,the “wild-type” F(ab)-phage, p426 was used as the template forconstruction of library-specific “stop” templates. By introducing stopcodons (TAA or TGA), the original molecule is rendered inactive, therebyreducing background effects and template-specific (hybridization) biasin the mutagenesis steps for constructing the library (Lowman & Wells,Methods: Comp. Methods Enzymol. 3: 205 (1991)). These templates wereconstructed using single-stranded template-directed mutagenesis (Kunkelet al., Methods Enzymol. 204: 125 (1991)), with the oligonucleotideslisted in Table 10.

[0469] Subsequently, these stop-templates were used in a second round ofmutagenesis, using the oligos listed in Table 11, to generate librariesin each of the indicated CDR regions. NNS degenerate codons were used toyield all twenty amino acids in each of the indicated CDR regions.(Nucleotide bases are indicated in single-letter IUPAC nomenclature;N=A, G, C or T; S=G or C). NNS degenerate codons were used to yield alltwenty amino acids at each randomized positions, using 32 differentpossible codons. An amber stop codon (TAG) encodes Gln in the suppressorsystem used here; i.e., the supE suppressor strain XL-1 Blue; Bullock etal. Biotechniques 5: 376 (1987). The presence of an amber codon betweenthe heavy-chain antibody domain and the g3p domain on phage permits theexpression of the phage-displayed fusion protein only in ambersuppressor strains of E. coli, while soluble F(ab) protein can beobtained with this same construct in non-suppressor strains of E. coli.(Lowman et al. Biochemistry 30: 10832 (1991); Lowman and Wells, MethodsComp. Methods. Enzymol. 3: 205 (1991); Hoogenboom et al., Nucl. AcidsRes. 19: 4133 (1991). However, other stop codons for use in other E.coli phage expression systems are apparent to those of ordinary skill inthe art.

[0470] The products of the random mutagenesis reaction were transformedinto E. coli cells (Stratagene, XL-1 Blue) by electroporation andamplified by growing overnight at 37° C. with M13K07 helper phage(Vierra and Messing, Methods Enzymol 153: (1987)). TABLE 10Stop-Template Oligos for First-Round Mutagenesis Oligo sequence no.Region Sequence HL-208 VL1 ACC TGC CGT GCC AGT TAA TAA GTC TAA TAA GAAGGT GAT AGC TAC (SEQ ID NO:27) HL-209 VH3 GCC AGT CAG AGC GTC TAA TAATAA GGT TGA AGC TAC CTG AAC TGG T (SEQ ID NO:28) HL-210 VH3 TGT GCT CGAGGC AGC TAA TAA TAA GGT TAA TGG TAA TTC GCC GTG TGG GG (SEQ ID NO:29)HL-220 VL2 G AAA CTA CTG ATT TAC TAA TAA TAA TAA CTG GAG TCT GGA GTC(SEQ ID NO.30) HL-221 VL3 CT TAT TAC TGT CAG CAA AGT TAA TAA TAA CCG TAAACA TTT GGA CAG GGT (SEQ ID NO:31) ACC HL-222 VH1 G TCC TGT GCA GTT TCTTAA TAA TAA TAA TAA TCC GGA TAC AGC TGG (SEQ ID NO:32) HL-223 VH1 GCCTAC TCC ATC ACC TAA TAA TAA AGC TGA AAC TGG ATC CGT CAG (SEQ ID NO:33)HL-224 VH2 GG GTT GCA TCG ATT TAA TAA TAA GGA TAA ACT TAA TAT AAC CCTAGC CTC AAG (SEQ ID NO:34) HL-225 VL1 AAG CCG GTC GAC AGG TAA TAA GATTAA TAC TAA AAC TGG TAT CAA CAG (SEQ ID NO:35)

[0471] TABLE 11 Library-Specific, Degenerate Oligos for Second RoundMutagenesis HL-212 VL1 ACC TGC CGT GCC AGT NNS NNS GTC NNS NNS GAA GGTGAT AGC TAC (SEQ ID NO:36) HL-213 VH3 GCC AGT CAG AGC GTC NNS NNS NSSGGT NNS AGC TAC CTG AAC TGG (SEQ ID NO:37) HL-214 VH3 TGT GCT CGA GGCAGC NNS NNS NNS GGT NNS TGG NNS TTC GCC GTG TGG GG (SEQ ID NO:38) HL-231VL2 G AAA CTA CTG ATT TAC NNS NNS NNS NNS CTG GAG TCT GGA GTC (SEQ IDNO:39) HL-232 VL3 CT TAT TAC TGT CAG CAA AGT NNS NNS NNS CCG NNS ACA TTTGGA CAG GGT ACC (SEQ ID NO:40) HL-233 VH1 G TCC TGT GCA GTT TCT NNS NNSNNS NNS NNS TCC GGA TAC AGC TGG (SEQ ID NO:41) HL-234 VH1 GTT TCT GGCTAC TCC ATC ACC NNS NNS NNS AGC NNS AAC TGG ATC CGT CAG (SEQ ID NO:42)HL-235 VH1 GG GTT GCA TCG ATT NNS NNS NNS GGA NNS ACT NNS TAT AAC CCTAGC GTC AAG (SEQ ID NO:43) HL-236 VL1 AAG CCG GTC GAC AGG NNS NNS GATNNS TAC NNS AAC TGG TAT CAA CAG (SEQ ID NO:44)

[0472] II. Phage Binding Selections.

[0473] For affinity-selections of phage particles displaying F(ab)variants, phage were prepared by sodium chloride/polyethylene glycol(NaCl/PEG) precipitation from E. coli culture supernatants. The phagewere suspended in PBS buffer, then diluted into horse serum (catalog no.A-3 311-D, Hyclone, Logan, Utah) containing 0.05% Tween™-20, as well asa non-displaying phage as a negative control. As a positive control,“wild-type” E426 F(ab)-phage were mixed with non-displaying phage andsubjected to mock-selections.

[0474] Maxisorp 96-well plastic plates (Nunc) were coated with 2 μg/mlIgE (human IgE; Genentech lot #9957-36) in 50 mM sodium carbonatebuffer, pH 9.6, overnight at 4° C. The IgE solution was then removed,and the plates were incubated with a blocking solution of horse serum(without Tween™-20), for 2 hours at ambient temperature.

[0475] The blocking solution was removed, and the phage solution wasincubated on the plates for 1 hour at room temperature. Thereafter, thephage solution was removed and the plates washed 10 times with PBS/Tweenm⁻²⁰ (0.05%) buffer. The wells were filled with PBS/Tween and allowed toincubate for another 10 minutes, after which the plates were againwashed 10 times.

[0476] F(ab)-phage remaining bound to the plate were eluted with 20 mMHCl, neutralized with Tris-HCl, pH 8, and propagated with helper phageas described above. An aliquot of phage was serially diluted, mixed withfresh XL-1 Blue cells, plated onto appropriate antibiotic plates, andthe number of CFUs (colony-forming units) of F(ab)-displaying(carbenicilin-resistant; CFUa) or non-displaying(chloramphenicol-resistant; CFUc) eluted phage were counted. Theenrichment (Emut) of F(ab)-displaying over non-displaying phage at eachround was calculated as (CFUa/CFUc) for the eluted pool divided by(CFUa/CFUc) for the starting pool. The enrichment for the wild-typecontrol phage (Ewt) was calculated in the same way.

[0477] Subsequent rounds of affinity selections were carried out asdescribed above, except that the incubation period following the first10 washes was increased in each round. In order to compare theefficiency of phage selection from round to round under increasingstringency conditions, the enrichment factor at each round wasnormalized to that of the wild-type control. The ratio of bindingenrichment for each pool to that of the wild-type (Emut/Ewt) is shown inFIG. 6. Since at equilibrium a greater fraction of a high-affinityvariant should be bound to the IgE plate than of a lower affinityvariant, higher-affinity variants should be recovered more efficiently,and therefore display greater relative enrichments. Indeed, the VL1libraries showed successively improved relative enrichments, up to about10-fold greater relative enrichments than wild-type after 5-6 rounds ofselection. By this measure, VL1 libraries showed greater improvement inaffinity over wild-type than did the VH3 libraries. The disparity inresults between the two sets of CDR libraries could reflect a greaterenergetic contribution to antigen binding by VL1. Alternatively, the VH3CDR of E25 may be already more nearly optimized for IgE binding than theVL1 CDR, thus permitting a greater relative improvement in the bindinginteractions contributed by VL I through sidechain substitutions.

[0478] DNA sequencing showed that most F(ab)-phage variants from thefirst VL CDR1 library (randomizing positions 27, 28, 39 and 31) hadconserved the wild-type residue D30, and preferentially mutated Y31 G(Table 12, wherein clones from round 3 are designated by 212-3.x, andthose from round 6 are designated 212-6.x). Although a variety ofsubstitutions were observed at positions Q27 and S28, one clone,containing Q27K and S28P, dominated the phage pool after 6 rounds ofselection. This clone also contained the preferred residues D30 and G31,suggesting that this combination of sidechains might be optimal forIgE-binding.

[0479] In the second VL CDR1 library (randomizing positions 30, 31, 32and 34), most selectants conserved wild-type residues at D30 and E32;only the wild-type D34 was observed among the sequenced clones. In thislibrary, a variety of residue types was observed at Y31. An additional,spurious mutation, G33S, was observed in two clones, 213-6.7 and 213-6.8(Table 12).

[0480] Sequencing analysis of clones from the VH CDR3 library after 3rounds of selection showed that the library had essentially converged toa single clone, i.e., 214-3.1, having wild-type residues at positions101-103, with substitutions H105T and H107Y (Table 12).

[0481] III. Phage-ELISA Assays of Selected F(ab) Clones.

[0482] To evaluate the results of the phage-binding selections, phagewere transfected into E. coli XL-1 Blue cells and propagated in liquidculture, or plated onto antibiotic containing plates. Clones-wererandomly picked from these plates for sequencing and binding analysis bycompetitive-phage-ELISA. (Cunningham et al, EMBO J. 13: 2508 (1994);Lowman, Chapter 24, in Methods in Molecular Biology, vol. 87, S. Cabilly(ed.), Humana Press Inc., Totawa, N.J. (1997).

[0483] To evaluate the relative IgE binding affinities, phage weretitrated on a plate coated with IgE as described above to normalize thedisplayed F(ab) concentrations. Phage were pre-mixed with serialdilutions of IgE, then added to an IgE-coated plate, and incubated for 1hour at room temperature. The plates were then washed ten times withPBS/Tween, and a solution of rabbit anti-phage antibody mixed with agoat-anti-rabbit conjugate of horseradish peroxidase was added. After 1hour incubation at room temperature, the plates were developed with achromogenic substrate, o-phenylenediamine (Sigma). The reaction wasstopped with addition of 1/2 volume of 2.5 M H₂SO₄ Optical density at490 nm was measured on a spectrophotometric plate reader. The IC50 ofeach variant was determined by fitting a 4-parameter curve to each dataset (Lowman, Methods in Mol. Biol., supra). The relative bindingaffinity of each cloned phage variant was determined as the ratio of itsIC50 to that of the starting phage, E426 (Tables 12-13).

[0484] In some cases, phage pools from a given round of selection weretested en masse in order to obtain an estimate of the populationaveraged relative affinity [IC50(wt)/IC50(mutant)] for IgE. For example,the VL CDR1 library, residues 32, 33, 35 and 37 showed only 3.6-foldimproved affinity versus E426 after 5 rounds of selection, eventhoughthe parental variant of this library (E26) appeared to have 25-foldimproved affinity. Therefore, the VL-CDR1 library of these particularresidues was not pursued further. On the other hand, the VH CDR2 phagepool showed 6.2 fold improved affinity over its parental e426 phage.

[0485] Phage libraries were also created of CDR domains VL CDR2,residues 54-57 and VL CDR3, residues 96-98, 99 and 100. However, aminoacid substitutions at these positions failed to generate any enrichmentover e426. A phage library generated for VH CDR1, residues 26-30 alsofailed to generate any enrichment over E26, and was found to bedominated by contaminating E26-phage. This suggests that no variants ofhigher affinity than E26 were present in the initial libraries.

[0486] Phage-libraries of CDR domains VL CDR1, residues 27, 28, 30, 31,32, 34 as well as VH CDR1, residues 101, 102, 103, 105 and 107 arereported in Table 12, while VH CDR2 is reported in Table 13. In Tables12 and 13, clone libraries which did not indicate affinity appreciablegreater that of E26 were not pursued further and the binding improvementfactor was not determined. TABLE 12 F(ab)-Phage Clones from IgE BindingSelections Fold improved binding phage VL CDR1 residue VH CDR3 residue(phage clone 27 28 30 31 32 34 101 102 103 105 107 ELISA) E426 Q S D Y ED H Y F H H -I- 212-3.1 M R Y G — — — — — — — not (x2) determined212-3.2 A Y N G — — — — — — — 3.5 212-3.3 G G Y G — — — — — — — 6.9212-3.5 M G E A — — — — — — — not determined 212-6.1 E Q D W — — — — — —— 23 212-6.2 E R E S — — — — — — — not determined 212-6.4 E H D W — — —— — — — 23 212-6.5 S N S G — — — — — — — not determined 212-6.6 K E D S— — — — — — — not determined 212-6.7 K P D G — — — — — — — 25 (x8) (E26)212-6.15 R P D T — — — — — — — not determined 212-6.16 R S D G — — — — —— — not determined 212-6.17 V T H S — — — — — — — not determined 213-3.1— — D D C D — — — — — not determined 213-3.2 — — H D S D — — — — — notdetermined 213-3.3 — — D W Q D — — — — — 8.8 213-3.4 — — G D H D — — — —— 3.7 213-6.1 — — E R W D — — — — — not determined 213-6.3 — — D T E D —— — — — 14 (x2) 213-6.4 — — D W E D — — — — — 20 213-6.7 — — H N E D — —— — — not G33S determined 213-6.8 — — Y S N D — — — — — 14 G33S 213-6.9— — W G E D — — — — — not determined 213-6.11 — — Y S E D — — — — — notdetermined 213-6-12 — — E R D D — — — — — not determined 213-6.13 — — HE E D — — — — — not determined 213-6.14 — — D K K D — — — — — notdetermined 213-6.15 — — D R Q D — — — — — 15 214-3.1 — — — — — — H Y F TY 2.7 (x5) 214-3.6 — — — — — — H Y F S R not determined

[0487] TABLE 13 VH CDR2 Phage Clones VH CDR2 residue Phage clone 53 5455 57 59 fold improved binding E426 T Y D S N -1- 235-5.1 K Y S E K notdetermined* 235-5.2 K W H E M not determined* 235-5.3 K W W E A notdetermined* 235-5.4 H Y A R K not determined* 235-5.5 K Y H G A notdetermined*

[0488] IV. Combined Mutations From Phage Screening.

[0489] Mutations at different sites within protein often displayadditive effect upon protein function (Wells, Biochemistry 29: 8509(1990). Therefore, several mutations from the initial phage librariesdescribed above were combined to improve the binding to IgE. In order toreduce the probability of increasing immunogenicity of the anti-IgEantibody, the extent of mutations from E25 needed to be minimized. As aresult, only the mutations from the phage variants which displayed thegreatest improvement in affinity when measured independently were used.In addition, the frequency with which a given phage clone was observedmay be related to expression level and/or proteolytic stability (Lowman& Wells, 1991, supra). One particular clone from the VL1 library,212-6.7- renamed E26, was chosen because it exhibited an affinity25-fold improved over E426 in phage-ELISA assays (Table 14).

[0490] The VH CDR2 library also showed affinity improvements over E426,although such improvement was only measured to be 6.2 fold as measuredfor the pooled phage. The pooled phage affinity demonstrated improvedbinding affinity for at least some members of the pool without having tomeasure the affinity of all the individual members. Use of the pooledphage also permits the identification of how much affinity enhancementhas been obtained after a given round, and whether or not affinityselections should be continued (i.e. once a pool affinity has reached amaximum, subsequent rounds unlikely to confer additional enrichment). Assuch, use of pooled affinity data is a highly useful screening tool.

[0491] It was apparent that mutations in the VH CDR2 region couldfunction additively with those in VL CDR1 because the VH CDR2 loop liesdistant from the VL CDR1 loop in both crystal structure and molecularmodels. However, because some combinations of these mutations mightnevertheless be incompatible, we tested four different combinationmutants: E26 combined with the mutations found in clones 235-5.1,235-5.2, 235-5.3, and 235-5.4 (Table 14). These constructs were made byKunkel mutagenesis (Kunkel et al., Methods Enzymol. 204: 125 (1991))using the E26 F(ab)-phage as a template, with mutagenic oligos encodingthe VH2 mutations.

[0492] Phage-ELISA assays (Lowman, Methods in Molecular Biology, vol 87,Cabilly (ed.), Humana Press Inc., Totawa, N.J. (1997)) were used tocompare the final variants from combinations of the VL CDR1 mutations ine26 with the VH CDR2 mutations in clones 235-5.1, 235-5.2, 235-5.3 and235-5.4. Soluble F(ab) proteins were also prepared and compared in abiotin-IgE plate assay, reported below in Table 14 and in FIG. 7. TABLE14 relative affinity (fold F(ab) fragment IC₅₀ (nm) improved) E426 1.5-1- E26 0.17 8.9 E27 (E26 + 235 − 5.1) 0.040 38 E695 (E26 + 235 − 5.2)0.050 31 E696 (E26 + 235 − 5.3) 0.063 24 E697 (E26 + 235 − 5.4) 0.066 23

[0493] V. Biotin Plate Assay (FcεRI-IgG Chimera Competition Assay).

[0494] Introduction: The purpose of this example is to compare howdifferent anti-IgE F(ab)s compete with an immobilized high affinity IgEreceptor IgG chimera for binding to biotinylated human IgE in solutionphase when anti-IgE F(ab) and biotin-IgE are added simultaneously to aplate coated with the IgE receptor chimera. As the anti-IgE F(ab)concentration increases, the amount of biotin IgE that can bind to thereceptor on the plate decreases resulting in a lower optical densityvalue as measured by the spectrophotometer.

[0495] Nunc maxisorp plates (catalog no. F96) were coated with 100ng/well of FcεRI-IgG (Haak-Frendscho et al., J. Immunol. 151, 352(1993), (Genentech, lot #2148-74 (6.4 mg/ml)) by aliquoting 100 μl of a1 μg/ml stock solution in 50 nm sodium carbonate buffer (pH 9.6)for 12to 24 hours at 4° C. Plates were washed 3 times with ELISA wash buffer(0.05% polysorbate 20 (Sigma) in PBS (pH 7.4)) and blocked by incubatingwith 200 μl] ELISA assay buffer (Tris buffered saline, pH 4.45 with 0.5%RIA grade bovine serum albumin, Sigma; 0.05% polysorbate 20 and 4 mMEDTA) for 60 minutes. Following 3 washes with wash buffer, 100 μl ofserial 2 fold dilutions of anti-IgE F(ab)s in assay buffer at an initialconcentration of 200 nM were added to the ELISA plate in triplicate.Dilutions were performed with a Titertek® multichannel pipet.Biotinylated IgE in assay buffer (100 μl, 1/500 dilution of 0.5 mg/mlstock) was added to all wells and the mixture was incubated on aminiorbital shaker (Bellco) for 60 minutes at 25° C. IgE was affinitypurified from U266B1 myeloma (ATCC TIB 196) culture supernatant andbiotinylated using biocytin hydrazide (O'Shannessy et al., Immunol.Lett. 8: 273 (1984); Pierce Chemical). The samples were washed 5× withwash buffer, and the bound IgE was detected with 100 μlperoxidase-conjugated streptavidin (Zymed) at 1:3000 for 90 minutes. Thesamples were then washed again 6× with wash buffer followed by additionof 100 μl of substrate solution (400 μg/ml o-phenylenediaminedihydrochloride and 4 mM H₂O₂ in PBS), and incubated for 6 minutes. Thereaction was then stopped with 4.5 M H₂SO₄ (100 μl) and the absorbanceread at 490 nm on a Uvmax microplate reader (Molecular Devices). Theabsorbance at various F(ab) concentration levels of E25, E26 and E27F(ab) antibody fragments are plotted in FIG. 8.

Conclusion

[0496] The plots in FIG. 8 indicate that both E26 and E27 have greateraffinity than E25 for the high affinity receptor and that E27 showed thegreatest affinity.

[0497] VI. BIAcore Assays of Soluble F(ab) Proteins.

[0498] The receptor-binding affinities of several F(ab) fragments werecalculated (Lofas & Johnson, J. Chem. Soc. Commun. 21, 1526-1528 (1990))from association and dissociation rate constants measured using aBIAcore™-2000 surface plasmon resonance system (BIAcore, Inc.). Abiosensor chip was activated for covalent coupling of IgE usingN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the manufacturer's (BIAcore)instructions. IgE was diluted into 10 nM sodium acetate buffer (pH 4.5)which was further diluted to approximately 30 μg/ml and injected overthe chip to obtain a signal of 800 to 12,400 response units (RU) ofimmobilized material. Since the signal in RU is proportional to the massof immobilized material this represents a range of immobilized IgEdensities on the matrix of about 0.4 to 6.5 pmol/cm². Finally, 1Methanolamine was injected as a blocking agent. Regenerations werecarried out with 4.5 M MgCl₂.

[0499] For kinetics measurements, 1.5 serial dilutions of F(ab) antibodyfragments were injected over the IgE chip in PBS/Tween buffer (0.05%Tween-20 in phosphate buffered saline) at 25° C. using a flow rate of 20pl/min. [FIG. 9].

[0500] Dissociation data were fit to a one-site model to obtain koff+/−s.d. (standard deviation of measurements). Pseudo-first order rateconstant (ks) were calculated for each association curve, and plotted asa function of protein concentration to obtain kon +/−s.e. (standarderror of fit). Equilibrium dissociation constants for Fab:IgE binding,Kd's, were calculated from SPR measurements as koff/kon. In the absenceof experimental artefacts, such as re-binding of dissociated F(ab), theobserved off-rate is independent of F(ab) concentration. Also, since theequilibrium dissociation constant, Kd, is inversely proportional tokoff, an estimate of affinity improvement can be made assuming theassociation rate (kon) is a constant for all variants. The off-rates,along with calculated half-life of dissociation, are displayed in Table15. TABLE 15 Dissociation Kinetics F(ab) K_(off) × 10⁻⁴ (sec⁻¹) t_(1/2)(min) improved (fold) E25 22 ± 4  5.3 -1- E26 3.6 ± 0.2 41 7.7 E27(E26 + 235 − 5.1) 0.98 118 22 E695 (E26 + 235 − 5.2) 0.94 122 23 E696(E26 + 235 − 5.3) 1.4 83 16 E697 (E26 + 235 − 5.4) 1.5 77 15

[0501] VII. F(ab) Expression and Purification.

[0502] Anti-IgE F(ab) E25 (Presta et al., J. Immunol 151: 2623-2632(1993)) and variants in phagemids derived from p426 (FIG. 10) wereexpressed in E. coli strain 34B8. Toothpick cultures (10 ml) in 2YTmedia with 50 μg/ml carbenicillin were incubated 8 hours at 37° C. andthen transferred to 1 liter of modified AP-5 containing 50 μg/mlcarbenicillin and incubated for 24 hours at 37° C. Cultures werecentrifuged in 500 ml bottles at 7,000 rpm for 15 minutes at 4° C. Thepellet was frozen for at least 3 hours at −20° C. Each 500 ml pellet wassuspended in 12.5 ml cold 25% sucrose in 50 mM Tris pH 8.0 containing 1mM benzamidine (Sigma) at 4° C. Suspension was solubilized by stirringat 4° C. for 3 hours. Suspension was centrifuged at 18,000 rpm for 15minutes at 4° C. and the F(ab)s expressed in the supernatant werepurified by protein G (Pharmacia) affinity chromotography. The columnwas washed with a solution of 10 mM Tris (pH 7.6) and 1 mM EDTA (pH 8.0)and the F(ab)s were eluted with 2.5× column volumes of 100 mM aceticacid (pH 3.0) and immediately returned to neutral pH with 0.5 volumes of1M Tris pH 8.0. Eluates were concentrated and buffer exchanged againstPBS with centricon 30 microcentrators (Amicon). Protein concentrationwas determined by absorbance at 280 nM with a spectrophotometer (BeckmanDU 64) and sample purity was evaluated using 4-20% SDS PAGE gels (Novex)under reducing conditions with 5% β-mercaptoethanol.

[0503] VIII. Results and Conclusion.

[0504] The results of phage-ELISA competition experiments show thatwhile E26 F(ab)-phage was about 9-fold improved in affinity over E426,the combination variants E695, E696 and E697 were 20-40 fold improvedover E426-phage. Additional combinations of phage-derived mutationscould yield antibody variants with similarly improved affinities.

[0505] When F(ab) soluble proteins were tested in a biotin-IgE plateassay, E26 F(ab) and E27 F(ab) were about 10-fold and 30-fold improved,respectively, over E25, for inhibiting IgE binding to FcεRI-IgG. Theoff-rate determination by BIAcore analysis support these relativeaffinities. In particular, E26 and E27 showed 7.7 fold and 22-foldslower off-rates than E25. Longer half-lives imply that the IgE is“occupied” or rendered incapable of binding to the high affinityreceptor for a longer period, thus resulting in improved potency of theanti-IgE therapeutic.

[0506] Thus, both equilibrium and kinetic binding data support theconclusion that E26 and E27 F(ab)s bind IgE about 10-fold and 30-foldmore tightly, respectively, than E25. The full-length antibodies (IgGs)containing the corresponding F(ab) mutations are expected to displaysimilar relative affinities to E25 IgG.

1 44 1 6127 DNA Artificial Sequence Expression plasmid 1 gaattcaacttctccatact ttggataagg aaatacagac atgaaaaatc 50 tcattgctga gttgttatttaagcttgccc aaaaagaaga agagtcgaat 100 gaactgtgtg cgcaggtaga agctttggagattatcgtca ctgcaatgct 150 tcgcaatatg gcgcaaaatg accaacagcg gttgattgatcaggtagagg 200 gggcgctgta cgaggtaaag cccgatgcca gcattcctga cgacgatacg250 gagctgctgc gcgattacgt aaagaagtta ttgaagcatc ctcgtcagta 300aaaagttaat cttttcaaca gctgtcataa agttgtcacg gccgagactt 350 atagtcgctttgtttttatt ttttaatgta tttgtaacta gaattcgagc 400 tcggtacccg gggatcctctcgaggttgag gtgattttat gaaaaagaat 450 atcgcatttc ttcttgcatc tatgttcgttttttctattg ctacaaacgc 500 gtacgctgat atccagctga cccagtcccc gagctccctgtccgcctctg 550 tgggcgatag ggtcaccatc acctgccgtg ccagtcagag cgtcgattac600 gaaggtgata gctacctgaa ctggtatcaa cagaaaccag gaaaagctcc 650gaaactactg atttacgcgg cctcgtacct ggagtctgga gtcccttctc 700 gcttctctggatccggttct gggacggatt tcactctgac catcagcagt 750 ctgcagccag aagacttcgcaacttattac tgtcagcaaa gtcacgagga 800 tccgtacaca tttggacagg gtaccaaggtggagatcaaa cgaactgtgg 850 ctgcaccatc tgtcttcatc ttcccgccat ctgatgagcagttgaaatct 900 ggaactgctt ctgttgtgtg cctgctgaat aacttctatc ccagagaggc950 caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg 1000agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 1050 accctgacgctgagcaaagc agactacgag aaacacaaag tctacgcctg 1100 cgaagtcacc catcagggcctgagctcgcc cgtcacaaag agcttcaaca 1150 ggggagagtg ttaagctgat cctctacgccggacgcatcg tggccctagt 1200 acgcaagttc acgtaaaaag ggtatctaga ggttgaggtgattttatgaa 1250 aaagaatatc gcatttcttc ttgcatctat gttcgttttt tctattgcta1300 caaacgcgta cgctgaggtt cagctggtgg agtctggcgg tggcctggtg 1350cagccagggg gctcactccg tttgtcctgt gcagtttctg gctactccat 1400 cacctccggatacagctgga actggatccg tcaggccccg ggtaagggcc 1450 tggaatgggt tgcatcgattacgtatgacg gatcgactaa ctataaccct 1500 agcgtcaagg gccgtatcac tataagtcgcgacgattcca aaaacacatt 1550 ctacctgcag atgaacagcc tgcgtgctga ggacactgccgtctattatt 1600 gtgctcgagg cagccactat ttcggtcact ggcacttcgc cgtgtggggt1650 caaggaaccc tggtcaccgt ctcctcggcc tccaccaagg gcccatcggt 1700cttcccccta gcaccctcct ccaagagcac ctctgggggc acagcggccc 1750 tgggctgcctggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 1800 aactcaggcg ccctgaccagcggcgtgcac accttcccgg ctgtcctaca 1850 gtcctcagga ctctactccc tcagcagcgtggtgaccgtg ccctccagca 1900 gcttgggcac ccagacctac atctgcaacg tgaatcacaagcccagcaac 1950 accaaggtgg acaagaaagt tgagcccaaa tcttgtgaca aaactcacac2000 ctagagtggc ggtggctctg gttccggtga ttttgattat gaaaagatgg 2050caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta 2100 cagtctgacgctaaaggcaa acttgattct gtcgctactg attacggtgc 2150 tgctatcgat ggtttcattggtgacgtttc cggccttgct aatggtaatg 2200 gtgctactgg tgattttgct ggctctaattcccaaatggc tcaagtcggt 2250 gacggtgata attcaccttt aatgaataat ttccgtcaatatttaccttc 2300 cctccctcaa tcggttgaat gtcgcccttt tgtctttagc gctggtaaac2350 catatgaatt ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc 2400tttgcgtttc ttttatatgt tgccaccttt atgtatgtat tttctacgtt 2450 tgctaacatactgcgtaata aggagtctta atcatgccag ttcttttggc 2500 tagcgccgcc ctataccttgtctgcctccc cgcgttgcgt cgcggtgcat 2550 ggagccgggc cacctcgacc tgaatggaagccggcggcac ctcgctaacg 2600 gattcaccac tccaagaatt ggagccaatc aattcttgcggagaactgtg 2650 aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc2700 cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg 2750tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt 2800 tgccttactggttagcagaa tgaatcaccg atacgcgagc gaacgtgaag 2850 cgactgctgc tgcaaaacgtctgcgacctg agcaacaaca tgaatggtct 2900 tcggtttccg tgtttcgtaa agtctggaaacgcggaagtc agcgccctgc 2950 accattatgt tccggatctg catcgcagga tgctgctggctaccctgtgg 3000 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt3050 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt 3100tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct 3150 ctctcgtttcatcggtatca ttacccccat gaacagaaat tcccccttac 3200 acggaggcat caagtgaccaaacaggaaaa aaccgccctt aacatggccc 3250 gctttatcag aagccagaca ttaacgcttctggagaaact caacgagctg 3300 gacgcggatg aacaggcaga catctgtgaa tcgcttcacgaccacgctga 3350 tgagctttac cgcaggatcc ggaaattgta aacgttaata ttttgttaaa3400 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 3450aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg 3500 agtgttgttccagtttggaa caagagtcca ctattaaaga acgtggactc 3550 caacgtcaaa gggcgaaaaaccgtctatca gggctatggc ccactacgtg 3600 aaccatcacc ctaatcaagt tttttggggtcgaggtgccg taaagcacta 3650 aatcggaacc ctaaagggag cccccgattt agagcttgacggggaaagcc 3700 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta3750 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc 3800gcgcttaatg cgccgctaca gggcgcgtcc ggatcctgcc tcgcgcgttt 3850 cggtgatgacggtgaaaacc tctgacacat gcagctcccg gagacggtca 3900 cagcttgtct gtaagcggatgccgggagca gacaagcccg tcagggcgcg 3950 tcagcgggtg ttggcgggtg tcggggcgcagccatgaccc agtcacgtag 4000 cgatagcgga gtgtatactg gcttaactat gcggcatcagagcagattgt 4050 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga4100 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg 4150cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 4200 aatacggttatccacagaat caggggataa cgcaggaaag aacatgtgag 4250 caaaaggcca gcaaaaggccaggaaccgta aaaaggccgc gttgctggcg 4300 tttttccata ggctccgccc ccctgacgagcatcacaaaa atcgacgctc 4350 aagtcagagg tggcgaaacc cgacaggact ataaagataccaggcgtttc 4400 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc4450 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4500ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 4550 gctgtgtgcacgaacccccc gttcagcccg accgctgcgc cttatccggt 4600 aactatcgtc ttgagtccaacccggtaaga cacgacttat cgccactggc 4650 agcagccact ggtaacagga ttagcagagcgaggtatgta ggcggtgcta 4700 cagagttctt gaagtggtgg cctaactacg gctacactagaaggacagta 4750 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg4800 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4850tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4900 ttgatcttttctacggggtc tgacgctcag tggaacgaaa actcacgtta 4950 agggattttg gtcatgagattatcaaaaag gatcttcacc tagatccttt 5000 taaattaaaa atgaagtttt aaatcaatctaaagtatata tgagtaaact 5050 tggtctgaca gttaccaatg cttaatcagt gaggcacctatctcagcgat 5100 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa5150 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5200cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 5250 cggaagggccgagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5300 agtctattaa ttgttgccgggaagctagag taagtagttc gccagttaat 5350 agtttgcgca acgttgttgc cattgctgcaggcatcgtgg tgtcacgctc 5400 gtcgtttggt atggcttcat tcagctccgg ttcccaacgatcaaggcgag 5450 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct5500 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 5550ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 5600 ctgtgactggtgagtactca accaagtcat tctgagaata gtgtatgcgg 5650 cgaccgagtt gctcttgcccggcgtcaaca cgggataata ccgcgccaca 5700 tagcagaact ttaaaagtgc tcatcattggaaaacgttct tcggggcgaa 5750 aactctcaag gatcttaccg ctgttgagat ccagttcgatgtaacccact 5800 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg5850 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 5900cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 5950 atttatcagggttattgtct catgagcgga tacatatttg aatgtattta 6000 gaaaaataaa caaataggggttccgcgcac atttccccga aaagtgccac 6050 ctgacgtcta agaaaccatt attatcatgacattaaccta taaaaatagg 6100 cgtatcacga ggccctttcg tcttcaa 6127 2 121 PRTMus musculus 2 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys ProSer 1 5 10 15 Gln Ser Leu Ser Leu Ala Cys Ser Val Thr Gly Tyr Ser IleThr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys35 40 45 Leu Glu Trp Met Gly Ser Ile Thr Tyr Asp Gly Ser Ser Asn Tyr 5055 60 Asn Pro Ser Leu Lys Asn Arg Ile Ser Val Thr Arg Asp Thr Ser 65 7075 Gln Asn Gln Phe Phe Leu Lys Leu Asn Ser Ala Thr Ala Glu Asp 80 85 90Thr Ala Thr Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105Trp His Phe Ala Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 110 115 120Ser 3 121 PRT Artificial Sequence F(ab) sequence derived from MAE11 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25 30 SerGly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu GluTrp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr 50 55 60 Ala Asp SerVal Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr PheTyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr TyrCys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105 Trp His Phe Ala ValTrp Gly Gln Gly Thr Leu Val Thr Val Ser 110 115 120 Ser 4 121 PRT Homosapiens unsure 30, 104-108 unknown amino acid 4 Glu Val Gln Leu Val GluSer Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Xaa 20 25 30 Ser Asp Tyr Ala Met Ser TrpVal Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Val Ile SerAsn Gly Ser Asp Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe ThrIle Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Ser ArgPhe Phe Xaa Xaa 95 100 105 Xaa Xaa Xaa Asp Val Trp Gly Gln Gly Thr LeuVal Thr Val Ser 110 115 120 Ser 5 111 PRT Mus musculus 5 Asp Ile Gln LeuThr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Gln Arg AlaThr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp 20 25 30 Tyr Asp Gly Asp SerTyr Met Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Ile Leu LeuIle Tyr Ala Ala Ser Tyr Leu Gly Ser 50 55 60 Glu Ile Pro Ala Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Asn Ile His Pro Val GluGlu Glu Asp Ala Ala Thr Phe 80 85 90 Tyr Cys Gln Gln Ser His Glu Asp ProTyr Thr Phe Gly Ala Gly 95 100 105 Thr Lys Leu Glu Ile Lys 110 6 111 PRTArtificial Sequence F(ab) light chain sequence derived from MAE11 6 AspIle Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 GlyAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp 20 25 30 Tyr AspGly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala ProLys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser HisGlu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile Lys110 7 111 PRT Homo sapiens unsure 33-34 unknown amino acid 7 Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp ArgVal Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp 20 25 30 Ile Ser Xaa XaaSer Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala Pro Lys LeuLeu Ile Tyr Ala Ala Ser Ser Leu Glu Ser 50 55 60 Gly Val Pro Ser Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile Ser Ser LeuGln Pro Glu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Tyr Asn Ser LeuPro Tyr Thr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile Lys 110 8 114PRT Artificial Sequence Light chain sequence derived from MAE11 8 AspIle Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 GlyAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val Asp 20 25 30 Gly GluGly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala ProLys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser HisGlu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile LysArg Thr Val 110 9 114 PRT Artificial Sequence Light chain sequencederived from MAE11 9 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln SerVal Asp 20 25 30 Tyr Glu Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys ProGly 35 40 45 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser50 55 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 6570 75 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 80 8590 Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100105 Thr Lys Val Glu Ile Lys Arg Thr Val 110 10 114 PRT ArtificialSequence Light chain sequence derived from MAE11 10 Asp Ile Gln Leu ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val ThrIle Thr Cys Arg Ala Ser Gln Ser Val Asp 20 25 30 Tyr Asp Gly Asp Ser TyrMet Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala Pro Lys Leu Leu IleTyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val Pro Ser Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile Ser Ser Leu Gln ProGlu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser His Glu Asp Pro TyrThr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile Lys Arg Thr Val 11011 114 PRT Artificial Sequence Heavy chain sequence derived from MAE1111 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25 30 SerGly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu GluTrp Val Ala Ser Ile Lys Tyr Ser Gly Glu Thr Lys Tyr 50 55 60 Asn Pro SerVal Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr PheTyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr TyrCys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105 Trp His Phe Ala ValTrp Gly Gln Gly 110 12 114 PRT Artificial Sequence Heavy chain sequencederived from MAE11 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu ValGln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly TyrSer Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro GlyLys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr AsnTyr 50 55 60 Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 8085 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95 100105 Trp His Phe Ala Val Trp Gly Gln Gly 110 13 218 PRT ArtificialSequence Light chain sequence derived from MAE11 13 Asp Ile Gln Leu ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val ThrIle Thr Cys Arg Ala Ser Gln Ser Val Asp 20 25 30 Tyr Asp Gly Asp Ser TyrMet Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala Pro Lys Leu Leu IleTyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val Pro Ser Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile Ser Ser Leu Gln ProGlu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser His Glu Asp Pro TyrThr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile Lys Arg Thr Val AlaAla Pro Ser Val Phe 110 115 120 Ile Phe Pro Pro Ser Asp Glu Gln Leu LysSer Gly Thr Ala Ser 125 130 135 Val Val Cys Leu Leu Asn Asn Phe Tyr ProArg Glu Ala Lys Val 140 145 150 Gln Trp Lys Val Asp Asn Ala Leu Gln SerGly Asn Ser Gln Glu 155 160 165 Ser Val Thr Glu Gln Asp Ser Lys Asp SerThr Tyr Ser Leu Ser 170 175 180 Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys Val 185 190 195 Tyr Ala Cys Glu Val Thr His Gln Gly LeuSer Ser Pro Val Thr 200 205 210 Lys Ser Phe Asn Arg Gly Glu Cys 215 14451 PRT Artificial Sequence Heavy chain sequence derived from MAE11 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25 30 SerGly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu GluTrp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr 50 55 60 Asn Pro SerVal Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr PheTyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr TyrCys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105 Trp His Phe Ala ValTrp Gly Gln Gly Thr Leu Val Thr Val Ser 110 115 120 Ser Ala Ser Thr LysGly Pro Ser Val Phe Pro Leu Ala Pro Ser 125 130 135 Ser Lys Ser Thr SerGly Gly Thr Ala Ala Leu Gly Cys Leu Val 140 145 150 Lys Asp Tyr Phe ProGlu Pro Val Thr Val Ser Trp Asn Ser Gly 155 160 165 Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 170 175 180 Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val Pro Ser Ser 185 190 195 Ser Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro 200 205 210 Ser Asn Thr Lys ValAsp Lys Lys Val Glu Pro Lys Ser Cys Asp 215 220 225 Lys Thr His Thr CysPro Pro Cys Pro Ala Pro Glu Leu Leu Gly 230 235 240 Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg ThrPro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp ProGlu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln 305 310 315 Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys 320 325 330 Ala Leu Pro Ala ProIle Glu Lys Thr Ile Ser Lys Ala Lys Gly 335 340 345 Gln Pro Arg Glu ProGln Val Tyr Thr Leu Pro Pro Ser Arg Glu 350 355 360 Glu Met Thr Lys AsnGln Val Ser Leu Thr Cys Leu Val Lys Gly 365 370 375 Phe Tyr Pro Ser AspIle Ala Val Glu Trp Glu Ser Asn Gly Gln 380 385 390 Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp 395 400 405 Gly Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser Arg 410 415 420 Trp Gln Gln Gly AsnVal Phe Ser Cys Ser Val Met His Glu Ala 425 430 435 Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 445 450 Lys 15 218 PRTArtificial Sequence Light chain sequence derived from MAE11 15 Asp IleGln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly AspArg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val Asp 20 25 30 Gly Glu GlyAsp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala Pro LysLeu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val Pro Ser ArgPhe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile Ser SerLeu Gln Pro Glu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser His GluAsp Pro Tyr Thr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile Lys ArgThr Val Ala Ala Pro Ser Val Phe 110 115 120 Ile Phe Pro Pro Ser Asp GluGln Leu Lys Ser Gly Thr Ala Ser 125 130 135 Val Val Cys Leu Leu Asn AsnPhe Tyr Pro Arg Glu Ala Lys Val 140 145 150 Gln Trp Lys Val Asp Asn AlaLeu Gln Ser Gly Asn Ser Gln Glu 155 160 165 Ser Val Thr Glu Gln Asp SerLys Asp Ser Thr Tyr Ser Leu Ser 170 175 180 Ser Thr Leu Thr Leu Ser LysAla Asp Tyr Glu Lys His Lys Val 185 190 195 Tyr Ala Cys Glu Val Thr HisGln Gly Leu Ser Ser Pro Val Thr 200 205 210 Lys Ser Phe Asn Arg Gly GluCys 215 16 451 PRT Artificial Sequence Heavy chain sequence derived fromMAE11 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 510 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 2530 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr 50 55 60 AsnPro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys AsnThr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala ValTyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105 Trp His PheAla Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 110 115 120 Ser Ala SerThr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 125 130 135 Ser Lys SerThr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 140 145 150 Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 155 160 165 Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 170 175 180 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 185 190 195 Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 200 205 210 Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 215 220 225 Lys Thr HisThr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 230 235 240 Gly Pro SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile SerArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser ThrTyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 320 325 330 Ala Leu ProAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 335 340 345 Gln Pro ArgGlu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 350 355 360 Glu Met ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 365 370 375 Phe Tyr ProSer Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 380 385 390 Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 395 400 405 Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 410 415 420 Trp Gln GlnGly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 425 430 435 Leu His AsnHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 445 450 Lys 17 218PRT Artificial Sequence Light chain sequence derived from MAE11 17 AspIle Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 GlyAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val Asp 20 25 30 Gly GluGly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys Ala ProLys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln Ser HisGlu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu Ile LysArg Thr Val Ala Ala Pro Ser Val Phe 110 115 120 Ile Phe Pro Pro Ser AspGlu Gln Leu Lys Ser Gly Thr Ala Ser 125 130 135 Val Val Cys Leu Leu AsnAsn Phe Tyr Pro Arg Glu Ala Lys Val 140 145 150 Gln Trp Lys Val Asp AsnAla Leu Gln Ser Gly Asn Ser Gln Glu 155 160 165 Ser Val Thr Glu Gln AspSer Lys Asp Ser Thr Tyr Ser Leu Ser 170 175 180 Ser Thr Leu Thr Leu SerLys Ala Asp Tyr Glu Lys His Lys Val 185 190 195 Tyr Ala Cys Glu Val ThrHis Gln Gly Leu Ser Ser Pro Val Thr 200 205 210 Lys Ser Phe Asn Arg GlyGlu Cys 215 18 451 PRT Artificial Sequence Heavy chain sequence derivedfrom MAE11 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln ProGly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser IleThr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly35 40 45 Leu Glu Trp Val Ala Ser Ile Lys Tyr Ser Gly Glu Thr Lys Tyr 5055 60 Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 7075 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 110 115 120Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 125 130 135Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 140 145 150Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 155 160 165Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 170 175 180Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 185 190 195Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 200 205 210Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 215 220 225Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 320 325 330Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 335 340 345Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 350 355 360Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 365 370 375Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 380 385 390Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 395 400 405Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 410 415 420Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 425 430 435Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 445 450Lys 19 218 PRT Artificial Sequence Light chain F(ab) sequence derivedfrom MAE11 19 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala SerVal 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro ValAsp 20 25 30 Gly Glu Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly35 40 45 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 5055 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 7075 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 80 85 90Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100 105Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 110 115 120Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 125 130 135Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 140 145 150Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 155 160 165Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 170 175 180Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 185 190 195Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 200 205 210Lys Ser Phe Asn Arg Gly Glu Cys 215 20 229 PRT Artificial Sequence Heavychain F(ab) sequence derived from MAE11 20 Glu Val Gln Leu Val Glu SerGly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser CysAla Val Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp IleArg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile Thr TyrAsp Gly Ser Thr Asn Tyr 50 55 60 Asn Pro Ser Val Lys Gly Arg Ile Thr IleSer Arg Asp Asp Ser 65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His TyrPhe Gly His 95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu ValThr Val Ser 110 115 120 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuAla Pro Ser 125 130 135 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyCys Leu Val 140 145 150 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpAsn Ser Gly 155 160 165 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser 170 175 180 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser 185 190 195 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro 200 205 210 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro LysSer Cys Asp 215 220 225 Lys Thr His Thr 21 229 PRT Artificial SequenceHeavy chain F(ab) derived from MAE11 21 Glu Val Gln Leu Val Glu Ser GlyGly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys AlaVal Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile ArgGln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile Lys Tyr SerGly Glu Thr Lys Tyr 50 55 60 Asn Pro Ser Val Lys Gly Arg Ile Thr Ile SerArg Asp Asp Ser 65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu ArgAla Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr PheGly His 95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val ThrVal Ser 110 115 120 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaPro Ser 125 130 135 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly CysLeu Val 140 145 150 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer Gly 155 160 165 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuGln Ser 170 175 180 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer Ser 185 190 195 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro 200 205 210 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp 215 220 225 Lys Thr His Thr 22 248 PRT Artificial Sequence sFvsequence derived from MAE11 22 Glu Val Gln Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val SerGly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln AlaPro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly SerThr Asn Tyr 50 55 60 Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg AspAsp Ser 65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala GluAsp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser110 115 120 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly125 130 135 Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser140 145 150 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val155 160 165 Asp Gly Glu Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro170 175 180 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu185 190 195 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp200 205 210 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr215 220 225 Tyr Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln230 235 240 Gly Thr Lys Val Glu Ile Lys Arg 245 23 248 PRT ArtificialSequence sFv sequence derived from MAE11 23 Glu Val Gln Leu Val Glu SerGly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser CysAla Val Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp IleArg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile Lys TyrSer Gly Glu Thr Lys Tyr 50 55 60 Asn Pro Ser Val Lys Gly Arg Ile Thr IleSer Arg Asp Asp Ser 65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His TyrPhe Gly His 95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu ValThr Val Ser 110 115 120 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser GluGly Gly Gly 125 130 135 Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser LeuSer Ala Ser 140 145 150 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala SerLys Pro Val 155 160 165 Asp Gly Glu Gly Asp Ser Tyr Leu Asn Trp Tyr GlnGln Lys Pro 170 175 180 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala SerTyr Leu Glu 185 190 195 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly SerGly Thr Asp 200 205 210 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu AspPhe Ala Thr 215 220 225 Tyr Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr ThrPhe Gly Gln 230 235 240 Gly Thr Lys Val Glu Ile Lys Arg 245 24 218 PRTArtificial Sequence Light chain F(ab)′2 sequence derived from MAE11 24Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val Asp 20 25 30 GlyGlu Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Lys AlaPro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser 50 55 60 Gly Val ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 Thr Leu Thr IleSer Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 80 85 90 Tyr Cys Gln Gln SerHis Glu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100 105 Thr Lys Val Glu IleLys Arg Thr Val Ala Ala Pro Ser Val Phe 110 115 120 Ile Phe Pro Pro SerAsp Glu Gln Leu Lys Ser Gly Thr Ala Ser 125 130 135 Val Val Cys Leu LeuAsn Asn Phe Tyr Pro Arg Glu Ala Lys Val 140 145 150 Gln Trp Lys Val AspAsn Ala Leu Gln Ser Gly Asn Ser Gln Glu 155 160 165 Ser Val Thr Glu GlnAsp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 170 175 180 Ser Thr Leu Thr LeuSer Lys Ala Asp Tyr Glu Lys His Lys Val 185 190 195 Tyr Ala Cys Glu ValThr His Gln Gly Leu Ser Ser Pro Val Thr 200 205 210 Lys Ser Phe Asn ArgGly Glu Cys 215 25 233 PRT Artificial Sequence Heavy chain F(ab)′2sequence derived from MAE11 25 Glu Val Gln Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val SerGly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln AlaPro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly SerThr Asn Tyr 50 55 60 Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg AspAsp Ser 65 70 75 Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala GluAsp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His95 100 105 Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser110 115 120 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser125 130 135 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val140 145 150 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly155 160 165 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser170 175 180 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser185 190 195 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro200 205 210 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp215 220 225 Lys Thr His Thr Cys Pro Pro Cys 230 26 233 PRT ArtificialSequence Heavy chain F(ab)′2 sequence derived from MAE11 26 Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser LeuArg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr SerTrp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val AlaSer Ile Lys Tyr Ser Gly Glu Thr Lys Tyr 50 55 60 Asn Pro Ser Val Lys GlyArg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr Phe Tyr Leu GlnMet Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala ArgGly Ser His Tyr Phe Gly His 95 100 105 Trp His Phe Ala Val Trp Gly GlnGly Thr Leu Val Thr Val Ser 110 115 120 Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser 125 130 135 Ser Lys Ser Thr Ser Gly Gly ThrAla Ala Leu Gly Cys Leu Val 140 145 150 Lys Asp Tyr Phe Pro Glu Pro ValThr Val Ser Trp Asn Ser Gly 155 160 165 Ala Leu Thr Ser Gly Val His ThrPhe Pro Ala Val Leu Gln Ser 170 175 180 Ser Gly Leu Tyr Ser Leu Ser SerVal Val Thr Val Pro Ser Ser 185 190 195 Ser Leu Gly Thr Gln Thr Tyr IleCys Asn Val Asn His Lys Pro 200 205 210 Ser Asn Thr Lys Val Asp Lys LysVal Glu Pro Lys Ser Cys Asp 215 220 225 Lys Thr His Thr Cys Pro Pro Cys230 27 45 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 27 acctgccgtg ccagttaata agtctaataa gaaggtgata gctac 45 2846 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 28 gccagtcaga gcgtctaata ataaggttga agctacctga actggt 46 2950 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 29 tgtgctcgag gcagctaata ataaggttaa tggtaattcg ccgtgtgggg 5030 43 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 30 gaaactactg atttactaat aataataact ggagtctgga gtc 43 31 53DNA Artificial Sequence Stop-Template Oligos for First-Round Mutagenesis31 cttattactg tcagcaaagt taataataac cgtaaacatt tggacagggt 50 acc 53 3246 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 32 gtcctgtgca gtttcttaat aataataata atccggatac agctgg 46 3345 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 33 gcctactcca tcacctaata ataaagctga aactggatcc gtcag 45 3453 DNA Artificial Sequence Stop-Template Oligos for First-RoundMutagenesis 34 gggttgcatc gatttaataa taaggataaa cttaatataa ccctagcctc 50aag 53 35 48 DNA Artificial Sequence Stop-Template Oligos forFirst-Round Mutagenesis 35 aagccggtcg acaggtaata agattaatac taaaactggtatcaacag 48 36 45 DNA Artificial Sequence Library-specific, defenerateoligos for second-round mutagenesis 36 acctgccgtg ccagtnnsnn sgtcnnsnnsgaaggtgata gctac 45 37 45 DNA Artificial Sequence Library-specific,degenerate oligos for second-round mutagenesis 37 gccagtcaga gcgtcnnsnnsnssggtnns agctacctga actgg 45 38 50 DNA Artificial SequenceLibrary-specific, degenerate oligos for second-round mutagenesis 38tgtgctcgag gcagcnnsnn snnsggtnns tggnnsttcg ccgtgtgggg 50 39 43 DNAArtificial Sequence Library-specific, degenerate oligos for second-roundmutagenesis 39 gaaactactg atttacnnsn nsnnsnnsct ggagtctgga gtc 43 40 53DNA Artificial Sequence Library-specific, degenerate oligos forsecond-round mutagenesis 40 cttattactg tcagcaaagt nnsnnsnnsc cgnnsacatttggacagggt 50 acc 53 41 46 DNA Artificial Sequence Library-specific,degenerate oligos for second-round mutagenesis 41 gtcctgtgca gtttctnnsnnsnnsnnsnn stccggatac agctgg 46 42 51 DNA Artificial SequenceLibrary-specific, degenerate oligos for second-round mutagenesis 42gtttctggct actccatcac cnnsnnsnns agcnnsaact ggatccgtca 50 g 51 43 53 DNAArtificial Sequence Library-specific, degenerate oligos for second-roundmutagenesis 43 gggttgcatc gattnnsnns nnsggannsa ctnnstataa ccctagcgtc 50aag 53 44 48 DNA Artificial Sequence Library-specific, degenerate oligosfor second-round mutagenesis 44 aagccggtcg acaggnnsnn sgatnnstacnnsaactggt atcaacag 48

What is claimed is:
 1. A method of adjusting the affinity of apolypeptide to a target molecule, comprising: a) identifying aspartylresidues which are prone to isomerization; and b) substitutingalternative residues and screening the resulting mutants for affinityagainst the target molecule.
 2. The method of claim 1 wherein step b) isaffinity maturation using phage display.
 3. The method of claim 2wherein the polypeptide is an antibody.
 4. The method of claim 3 whereinthe antibody is an anti-IgE antibody and the target molecule is IgE. 5.The method of claim 4 wherein the antibody is the sequence indicated as“E25” in FIG.
 12. (SEQ ID NOS: 13-14).
 6. The method of claim 5 whereinthe residues substituted are variable light chain CDR1 residuesAsp32Glu, Gln27Lys and Ser28Pro.
 7. The method of claim 6 wherein theadditionally substituted residues are variable heavy chain CDR2 residuesThr53Lys, Asp55Ser, Ser57Glu and Asn59Lys.
 8. An antibody moleculecomprising an e26 sequence selected from the group consisting-of: F(ab)fragment (SEQ ID NOS: 19-20], sFv fragment (SEQ ID NO: 22) or F(ab)′₂(SEQ ID NOS: 24-25).
 9. An antibody molecule having a sequence which issubstantially identical to the sequence “e26” of FIG. 12 (SEQ ID NOS:15-16).
 10. An antibody molecule comprising an E27 sequence selectedfrom the group consisting of: F(ab) fragment, (SEQ ID NOS: 19 and 21),sFv fragment (SEQ ID NO:23) or F(ab)′₂ [SEQ ID NOS: 24 and 26).
 11. Anantibody molecule having a sequence which is substantially identical tothe sequence “E27” of FIG. 12 (SEQ ID NOS: 17-18).
 12. An improvedantibody or functional fragment thereof having improved ragweed-inducedhistamine release inhibition properties as a result of application ofthe method of claim
 1. 13. An improved antibody or functional fragmentthereof having improved ragweed-induced histamine release inhibitionproperties as a result of application of the method of claim
 2. 14. Anucleic acid molecule having a sequence encoding for an E26 antibodyfragment selected from the group consisting of: F(ab), sFv and F(ab′)₂.15. A nucleic acid molecule having a sequence substantially identical toone encoding for E26. 16 A nucleic acid molecule having a sequenceencoding for an E27 antibody fragment selected from the group consistingof: F(ab), sFv and F(ab′)₂.
 17. A nucleic acid molecule having asequence substantially identical to one encoding for E27.
 18. A nucleicacid molecule which encodes for an antibody having improvedragweed-induced histamine release properties as a result of theapplication of the method of claim
 1. 19. A nucleic acid molecule whichencodes for an antibody having improved ragweed-induced histaminerelease properties as a result of the application of the method of claim2.
 20. A composition comprising pharmaceutically-acceptable excipient(s)in admixture with an E26 antibody molecule having a sequence selectedfrom the group consisting of: F(ab) (SEQ ID NOS: 19-20); sFv (SEQ ID NO:22) and F(ab)′₂ (SEQ ID NOS: 24-25).
 21. A composition comprisingpharmaceutically-acceptable excipient(s) in admixture with an antibodyhaving a sequence substantially similar to “E26” of FIG. 12 (SEQ ID NOS:15-16).
 22. A composition comprising pharmaceutically-acceptableexcipient(s) in admixture with an E27 antibody molecule having asequence selected from the group consisting of: F(ab) (SEQ ID NOS: 19and 20); sFv (SEQ ID NO: 23) and F(ab)′₂ (SEQ ID NOS: 24 and 26).
 23. Acomposition comprising pharmaceutically-acceptable excipient(s) inadmixture with an antibody molecule having a sequence substantiallysimilar to “E27” of FIG. 12 (SEQ ID NOS: 17-18).
 24. A method ofreducing or preventing the IgE mediated production of histamine in amammal comprising the administration of a therapeutically effectiveamount of an E26 antibody having a sequence selected from the groupconsisting of: F(ab) (SEQ ID NOS: 19-20); sFv (SEQ ID NO: 22) andF(ab′)₂ (SEQ ID NOS: 24-25).
 25. A method of reducing or preventing theIgE mediated production of histamine in a mammal comprising theadministration of a therapeutically effective amount of an antibodyhaving a sequence substantially similar to “e26” of FIG. 12 (SEQ ID NOS:15-16).
 26. A method of reducing or preventing the IgE mediatedproduction of histamine in a mammal comprising the administration of atherapeutically effective amount of an E27 antibody having a sequenceselected from the group consisting of: (SEQ ID NOS: 15-16).
 27. A methodof reducing or preventing the IgE mediated production of histamine in amammal comprising the administration of a therapeutically effectiveamount of an antibody having a sequence substantially similar to “E27”of FIG. 12 (SEQ ID NOS: 17-18).
 28. A method of treating a disordermediated by IgE comprising the administration to a mammal in needthereof a therapeutically effective amount of E26 antibody sequencefragment selected from the group consisting of: F(ab) (SEQ ID NOS:19-20); sFv (SEQ ID NO: 22) and F(ab′)₂ (SEQ ID NOS: 24-25).
 29. Amethod of treating a disorder mediated by IgE comprising theadministration to a mammal in need thereof a therapeutically effectiveamount of an antibody having a sequence substantially similar to “E26”of FIG. 12 (SEQ ID NOS: 15-16).
 30. A method of treating a disordermediated by IgE comprising the administration to a mammal in needthereof a therapeutically effective amount of E27 antibody moleculehaving a sequence fragment selected from the group consisting of: (SEQID NOS: 15-16)
 31. A method of treating a disorder mediated by IgEcomprising the administration to a mammal in need thereof atherapeutically effective amount of antibody molecule having a sequencesubstantially similar to “E27” of FIG. 12 (SEQ ID NOS: 17-18).